Effect of L-carnitine on postischemic inhibition of protein synthesis in the rat brain.

The purpose of this study was to investigate effects of carnitine administration on protein synthesis recovery after transient cerebral ischemia. Rats received L-carnitine in two doses of 16 mmol/kg i.p. 15 min before ischemia and just on the onset of reperfusion. Transient forebrain ischemia was induced by 4-vessel occlusion for 15 min, followed by 30 min or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. A protective effect was observed, on protein synthesis as well as the number of surviving neurons, in the L-carnitine-treated groups. Our results indicate that L-carnitine can exert a protective effect in the development of reperfusion-induced injury. L-carnitine significantly reduced the ischemia/reperfusion-induced inhibition of translation and neurodegeneration in the neocortex as well as in the highly sensitive hippocampus and dorsolateral striatum. We expect that the ability of L-carnitine to keep translational machinery on facilitates efficacy of postischemic remodulation of gene expression.

Glutathione transferase (GST) as a candidate molecular-based biomarker for soil toxin exposure in the earthworm Lumbricus rubellus.

The earthworm Lumbricus rubellus (Hoffmeister, 1843) is a terrestrial pollution sentinel. Enzyme activity and transcription of phase II detoxification superfamily glutathione transferases (GST) is known to respond in earthworms after soil toxin exposure, suggesting GST as a candidate molecular-based pollution biomarker. This study combined sub-proteomics, bioinformatics and biochemical assay to characterise the L. rubellus GST complement as pre-requisite to initialise assessment of the applicability of GST as a biomarker. L. rubellus possesses a range of GSTs related to known classes, with evidence of tissue-specific synthesis. Two affinity-purified GSTs dominating GST protein synthesis (Sigma and Pi class) were cloned, expressed and characterised for enzyme activity with various substrates. Electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) following SDS-PAGE were superior in retaining subunit stability relative to two-dimensional gel electrophoresis (2-DE). This study provides greater understanding of Phase II detoxification GST superfamily status of an important environmental pollution sentinel organism.

Increasing the function of the glutamate-nitric oxide-cyclic guanosine monophosphate pathway increases the ability to learn a Y-maze task.

N-methyl-D-aspartate (NMDA) receptors play a crucial role in learning. However, the molecular mechanisms by which NMDA receptors contribute to learning processes are not known in detail. Activation of NMDA receptors leads to increased calcium in the postsynaptic neuron. Calcium binds to calmodulin and activates neuronal nitric oxide synthase, increasing nitric oxide (NO), which activates soluble guanylate cyclase, increasing cGMP. Part of this cGMP is released to the extracellular space. Several reports indicate that impairment of this glutamate-NO-cGMP pathway reduces the ability to learn a Y-maze conditional discrimination task by rats. The aim of this work was to assess whether enhancing the function of this pathway increases the ability to learn this task. Prenatal exposure to the polybrominated diphenylether PBDE-99 during embryonic days 2-9 or 11-19 enhances the function of the glutamate-NO-cGMP pathway in cerebellum in vivo as assessed by microdialysis in freely moving rats. This was associated with an increase in the ability to learn the Y-maze task. Rats prenatally exposed to PBDE need fewer trials than control rats to learn the Y-maze task. These results show that the function of the glutamate-NO-cGMP modulates the ability of rats to learn the Y-maze task, that the function of the pathway under physiological conditions is not optimal for learning, and that performance in the Y-maze task may be improved by enhancing slightly the function of the pathway and cGMP formation.

Lymphocyte cytochrome c oxidase, cyclic GMP and cholinergic muscarinic receptors as peripheral indicators of carbon monoxide neurotoxicity after acute and repeated exposure in the rat.

Changes in cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) have been reported in rodents acutely exposed to carbon monoxide (CO). These endpoints measurable in lymphocytes may serve as peripheral markers of CO neurotoxicity. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was similarly affected both centrally and peripherally. Male Wistar rats either inhaled 500 ppm CO, 6 h/day, 5 days/week, 4 weeks (repeated exposure) or 2,400 ppm, 1 h (single exposure). Neither treatment altered brain or lymphocyte COX activity 1 and 7 days post-treatment. Also ineffective were repeated and acute CO treatments towards (3)H-quinuclidinyl benzilate (QNB) binding to MRs in cerebral cortex, hippocampus, striatum, cerebellum (respective controls, mean+/-S.D.: 171 +/- 45, 245 +/- 53, 263 +/- 14 and 77 +/- 7 fmol/mg protein) and lymphocytes (24 +/- 10 fmol/million cells) at the same time points. In lymphocytes control cGMP levels averaged 1.98 +/- 0.99 pmol/mg protein under basal conditions, and 3.94 +/- 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the CO-treated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after, compared to air-exposed rats. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NO-cGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain [Hernández-Viadel, M., Castoldi, A.F., Coccini, T., Manzo, L., Erceg, S., Felipo, V., 2004. In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum. Journal of Neurochemistry 89, 1,157-1,165], and supports the use of this peripheral endpoint as a biomarker of CO central effects.

Restoration of learning ability in hyperammonemic rats by increasing extracellular cGMP in brain.

Intellectual function is impaired in patients with hyperammonemia and hepatic encephalopathy. Chronic hyperammonemia with or without liver failure impairs the glutamate-nitric oxide-cGMP pathway function in brain in vivo and reduces extracellular cGMP in brain as well as the ability of rats to learn a Y maze conditional discrimination task. We hypothesized that the decrease in extracellular cGMP may be responsible for the impairment in learning ability and intellectual function and that pharmacological modulation of the levels of cGMP may restore learning ability. The aim of this work was to try to reverse the impairment in learning ability of hyperammonemic rats by pharmacologically increasing extracellular cGMP in brain. We assessed whether learning ability may be restored by increasing extracellular cGMP in brain by continuous intracerebral administration of: (1) zaprinast, an inhibitor of the phosphodiesterase that degrades cGMP or (2) cGMP. We carried out tests of conditional discrimination learning in a Y maze with control and hyperammonemic rats treated or not with zaprinast or cGMP. Learning ability was reduced in hyperammonemic rats, which needed more trials than control rats to learn the task. Continuous intracerebral administration of zaprinast or cGMP restored the ability of hyperammonemic rats to learn this task. Pharmacological modulation of extracellular cGMP levels in brain may be a useful therapeutic approach to improve learning and memory performance in individuals in whom cognitive abilities are impaired by different reasons, for example in patients with liver disease who present hyperammonemia and decreased intellectual function.

Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunts.

Patients with liver disease with overt or minimal hepatic encephalopathy show impaired intellectual capacity. The underlying molecular mechanism remains unknown. Rats with portacaval anastomosis or with hyperammonemia without liver failure also show impaired learning ability and impaired function of the glutamate-nitric oxide-cyclic guanine monophosphate (glutamate-NO-cGMP) pathway in brain. We hypothesized that pharmacological manipulation of the pathway in order to increase cGMP content could restore learning ability. We show by in vivo brain microdialysis that chronic oral administration of sildenafil, an inhibitor of the phosphodiesterase that degrades cGMP, normalizes the function of the glutamate-NO-cGMP pathway and extracellular cGMP in brain in vivo in rats with portacaval anastomosis or with hyperammonemia. Moreover, sildenafil restored the ability of rats with hyperammonemia or with portacaval shunts to learn a conditional discrimination task. In conclusion, impairment of learning ability in rats with chronic liver failure or with hyperammonemia is the result of impairment of the glutamate-NO-cGMP pathway. Moreover, chronic treatment with sildenafil normalizes the function of the pathway and restores learning ability in rats with portacaval shunts or with hyperammonemia. Pharmacological manipulation of the pathway may be useful for the clinical treatment of patients with overt or minimal hepatic encephalopathy.

In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum.

Carbon monoxide induces delayed neurological and neuropathological alterations, including memory loss and cognitive impairment. The bases for the delay remain unknown. Activation of soluble guanylate cyclase by nitric oxide modulates some forms of learning and memory. Carbon monoxide binds to soluble guanylate cyclase, activating it but interfering with its activation by nitric oxide. The aim of this work was to assess whether exposure of rats to carbon monoxide alters the activity of soluble guanylate cyclase or its modulation by nitric oxide in cerebellum or cerebral cortex. Rats exposed chronically or acutely to carbon monoxide were killed 24 h or 7 days later. Acute carbon monoxide exposure decreased cyclic guanosine monophosphate (cGMP) content and reduced activation of soluble guanylate cyclase by nitric oxide. Cortex was more sensitive than cerebellum to chronic exposure, which reduced activation of soluble guanylate cyclase by nitric oxide in cortex. In cerebellum, chronic exposure induced delayed impairment of soluble guanylate cyclase activation by nitric oxide. Acute exposure effects were also stronger at 7 days than at 24 h after exposure. This delayed impaired modulation of soluble guanylate cyclase by nitric oxide may contribute to delayed memory loss and cognitive impairment in humans exposed to carbon monoxide.

Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide.

Acute intoxication with large doses of ammonia leads to rapid death. The main mechanism for ammonia elimination in brain is its reaction with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase and consumes ATP. In the course of studies on the molecular mechanism of acute ammonia toxicity, we have found that glutamine synthetase activity and glutamine content in brain are modulated by NMDA receptors and nitric oxide. The main findings can be summarized as follows. Blocking NMDA receptors prevents ammonia-induced depletion of brain ATP and death of rats but not the increase in brain glutamine, indicating that ammonia toxicity is not due to increased activity of glutamine synthetase or formation of glutamine but to excessive activation of NMDA receptors. Blocking NMDA receptors in vivo increases glutamine synthetase activity and glutamine content in brain, indicating that tonic activation of NMDA receptors maintains a tonic inhibition of glutamine synthetase. Blocking NMDA receptors in vivo increases the activity of glutamine synthetase assayed in vitro, indicating that increased activity is due to a covalent modification of the enzyme. Nitric oxide inhibits glutamine synthetase, indicating that the covalent modification that inhibits glutamine synthetase is a nitrosylation or a nitration.Inhibition of nitric oxide synthase increases the activity of glutamine synthetase, indicating that the covalent modification is reversible and it must be an enzyme that denitrosylate or denitrate glutamine synthetase.NMDA mediated activation of nitric oxide synthase is responsible only for part of the tonic inhibition of glutamine synthetase. Other sources of nitric oxide are also contributing to this tonic inhibition. Glutamine synthetase is not working at maximum rate in brain and its activity may be increased pharmacologically by manipulating NMDA receptors or nitric oxide content. This may be useful, for example, to increase ammonia detoxification in brain in hyperammonemic situations.

Chronic hyperammonemia alters protein phosphorylation and glutamate receptor-associated signal transduction in brain.

There is substantial evidence that hyperammonemia is one of the main factors contributing to the neurological alterations found in hepatic encephalopathy. The mechanisms by which chronic moderate hyperammonemia affects brain function involves alterations in neurotransmission at different steps. This article reviews the effects of hyperammonemia on phosphorylation of key brain proteins involved in neurotransmission (the microtubule-associated protein (MAP-2), Na+/K+-ATPase and NMDA receptors). The physiological function of these proteins is modulated by phosphorylation and its altered phosphorylation in hyperammonemia may contribute to impairment of neurotransmission. The effects of chronic hyperammonemia on signal transduction pathways associated to glutamate receptors, such as the glutamate-nitric oxide (NO)-cGMP pathway, are also reviewed. The possible contribution of the impairment of this pathway in brain in vivo to the neurological alterations present in patients with hepatic encephalopathy is discussed.

Prevention of ammonia and glutamate neurotoxicity by carnitine: molecular mechanisms.

Carnitine has beneficial effects in different pathologies and prevents acute ammonia toxicity (ammonia-induced death of animals). Acute ammonia toxicity is mediated by excessive activation of the NMDA-type of glutamate receptors, which mediates glutamate neurotoxicity. We showed that carnitine prevents glutamate neurotoxicity in primary cultures of cerebellar neurons. This supports the idea that the protective effect of carnitine against ammonia toxicity is due to the protective effect against glutamate neurotoxicity. We are studying the mechanism by which carnitine protects against glutamate neurotoxicity. Carnitine increases the binding affinity of glutamate for metabotropic glutamate receptors. The protective effect of carnitine is lost if metabotropic glutamate receptors are blocked with specific antagonists. Moreover, activation of metabotropic glutamate receptors by specific agonists also prevents glutamate neurotoxicity. This indicates that the protective effect of carnitine against glutamate neurotoxicity is mediated by activation of metabotropic glutamate receptors. The molecule of carnitine has a trimethylamine group. Different compounds containing a trimethylamine group (carbachol, betaine, etc.) also prevent ammonia-induced animal death and glutamate-induced neuronal death. Moreover, metabotropic glutamate receptor antagonists also prevent the protective effect of most of these compounds. We summarize here some studies aimed to identify the mechanism and the molecular target that are responsible for the protective effect of carnitine against ammonia and glutamate neurotoxicity. Finally it is also shown that carnitine inhibits the hydrolysis of inositol phospholipids induced by activation of different types of metabotropic receptors, but this effect seems not responsible for its protective effects.