Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity.

AIMS/HYPOTHESIS: Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans. METHODS: Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy. RESULTS: Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules. CONCLUSIONS/INTERPRETATION: Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.

Early events and implication of F-actin and annexin I associated structures in the phagocytic uptake of Brucella suis by the J-774A.1 murine cell line and human monocytes.

Brucella spp. are facultative, intracellular pathogenic bacteria that cause brucellosis, a zoonosis affecting mammalian species. Brucella entry into myelomonocytic cell lines is highly enhanced by opsonization. Few studies have been undertaken to unravel the first interactions between these bacteria and their host cells. This paper deals with early events following contact of Brucella suis with the J-774A.1 phagocytic cell line and differentiated monocytes. Phagocytic uptake of bacteria was documented under a fluorescence microscope using GFP-expressing B. suis. Unlike entry in the J-774A. 1 cell line, non-opsonized Brucella entered differentiated human monocytes as efficiently as opsonized bacteria. However, following 1 h infections, a mean of only three bacteria were phagocytized and the whole monocyte population was only infected after a 4 h infection. Contact of non-opsonized Brucella with phagocytes did not induce marked structural changes at the cell surface, as revealed by scanning electron microscopy. Contact of Brucella (opsonized or not) elicited transient local recruitment of F-actin, revealed by phalloidin labelling, and of annexin I-associated structures, revealed by immunofluorescence staining. Finally, bacteria appeared to be rapidly internalized in monocytes once they had adhered to the cell surface. A low percentage of infected cells and few adhered and/or internalized bacteria following short-term infections could have resulted either from the fact that there were few sites of entry or the weak bacterial initial interactions with the host-cell membrane or the bacterial receptor.

Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis.

Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of Brucella virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D(3)-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the virB operon and manB), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of Brucella for the different factors identified are discussed.

A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis.

Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B. suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis. DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots. Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently. Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages. The virulence was restored by complementation with a plasmid containing the full virB region. The virB region appears to be essential for the intracellular survival and multiplication of B. suis.

Flupirtine, a nonopioid centrally acting analgesic, acts as an NMDA antagonist.

1. Flupirtine (Katadolon) is a member of a class of triaminopyridines and is used as a nonopioid analgesic agent with muscle relaxant properties. 2. In situ experiments have revealed that flupirtine protects against ischemic-induced insults to the retina and brain. 3. Data derived from in vitro and in vivo studies suggest that flupirtine functions as a weak N-methyl-D-aspartate (NMDA) antagonist with little evidence that it acts on AMPA-kainate type glutamate receptors. 4. No evidence could be found from binding studies to suggest that flupirtine has an affinity for any of the characterized binding sites associated with the NMDA receptor. 5. Studies on cultured cortical neurons show that the NMDA-induced influx of 45Ca2+ is more readily decreased by flupirtine when a reducing agent (dithiothreitol) is present. However, when N'-ethylmaleimide, which is thought to alkylate the NMDA receptor redox site, is present, no obvious effect on the NMDA-induced influx of 45Ca2+ is produced by flupirtine. 6. Flupirtine is also known to counteract the production of reactive oxygen species caused by ascorbate/iron as well as to prevent apoptosis in cells lacking NMDA receptors induced by oxidative stress. 7. To explain all the experimental data, it is suggested that flupirtine affects the redox state/pH/electrons in the cell. The specific way by which flupirtine antagonizes the NMDA receptor might be by an action on the known redox site of the receptor.

Melatonin protects primary cultures of rat cortical neurones from NMDA excitotoxicity and hypoxia/reoxygenation.

Studies on rat cortical cultures show that glutamate (10 microM) or hypoxia followed by reoxygenation causes damage to the cells as indexed by a release of lactate dehydrogenase (LDH). These effects could be counteracted by the N-methyl-D-aspartate (NMDA) antagonist MK-801 (2 microM) but not by the kainate/AMPA antagonist CNQX (100 microM). These data favour the view that the damage caused to the cells by glutamate and hypoxia/reperfusion is mediated via NMDA receptors. The damage to the cells could also be prevented by melatonin (100 microM). The melatonin effect is not mediated by specific receptors because it was not blunted by the melatonin antagonist, luzindole. Moreover, NMDA stimulated an accumulation of 45Ca2+ by cortical neurones, but although this effect was counteracted by MK-801, melatonin was ineffective, which showed that the neuroprotective effect of melatonin is not elicited by direct action with NMDA receptors. Ascorbate and iron stimulated the production of free radicals in a retinal cell preparation. Chelation of the iron with deferoxamine prevented this process as did melatonin while MK-801 had no effect. The combined findings suggest that melatonin counteracts the in vitro destructive effects of NMDA or hypoxia/reperfusion by preventing accumulation of excessive free radicals.

Induction of apoptosis in cultured human retinal pigment epithelial cells is counteracted by flupirtine.

PURPOSE: The aim of the study was to determine whether flupirtine can counteract the induction of apoptosis in cultured retinal pigment epithelium (RPE) cells. METHODS: Confluent cultures were subjected to experimental ischemia (medium free of serum, glucose, and oxygen) with or without various substances for specific periods. The cells were then examined for breakdown of DNA by the TUNEL procedure and agarose gel electrophoresis. Moreover cells were processed for the localization of oncogene proteins (bcl-2, TIAR, ICH-1t) associated with apoptosis. The effect of flupirtine on reactive oxygen species also was determined. RESULTS: When RPE cells were subjected to ischemia for 72 hours approximately 65% of cells remained attached to the coverslips and approximately 65% of their nuclei showed clear fragmentation of DNA by TUNEL. Most of the cells exhibited a shrunken appearance typical of apoptosis. Fragmentation of the DNA from cells given ischemia for 72 hours was also confirmed by agarose gel electrophoresis. Inclusion of flupirtine (flupirtine gluconate, 100 microM) or 10% fetal calf serum in the medium prevented ischemia-induced apoptosis occurring after 72 hours. Neither N-methyl-D-aspartate (NMDA) (100 microM) nondeferoxamine (100 microM) nor the NMDA antagonists dextromethorphan (100 microM), memantine (100 microM), and MK-801 (10 microM) had a similar effect. NMDA, and to a lesser extent memantine, induced apoptosis independently. Treatment of RPE cells in serum-free medium with flupirtine (flupirtine gluconate, 100 microM) for 72 hours caused an upregulation of bcl-2 protein. In contrast, the oncogene proteins for TIAR and ICH-1t, were lower in flupirtine-treated cells than in control cells. Flupirtine, like deferoxamine, prevents iron-ascorbate-induced reactive oxygen species formation in retinal cells, but only flupirtine prevents ischemia-induced apoptosis in RPE cells. CONCLUSIONS: The combined data demonstrate that flupirtine is an effective agent in preventing death by apoptosis. Flupirtine reduces formation of reactive oxygen species in retinal dissociates and causes changes in various oncogene products in RPE cultures, which may explain its action in preventing apoptosis induced by ischemia. The current results also suggest that NMDA receptors are not involved in the induction of ischemia-induced apoptosis in RPE, cells.

Retinal neurones containing kainate receptors are influenced by exogenous kainate and ischaemia while neurones lacking these receptors are not -- melatonin counteracts the effects of ischaemia and kainate.

The present experiments were carried out on three types of neurone in primary rabbit retinal cultures. One cell-type, bipolar neurones, have glutamate APB-type metabotropic receptors and can be identified by the presence of thetaPKC-immunoreactivity. The other two cell-types are primarily amacrine cells and can be 'stained' for the localisation of GABA immunoreactivity or for serotonin taken up from the medium. Most of the serotonin-accumulating and GABA-containing neurones contain glutamate kainate-type receptors. Exposure of the cultures to treatment of kainate (50 microM) or experimental ischaemia (8 h followed by 16 h reoxygenation) produced essentially similar findings. The serotonin-accumulating and GABA cells were affected as they were drastically reduced in numbers while the numbers of thetaPKC-containing cells were unaffected. Inclusion of the kainate/AMPA antagonist CNQX (100 microM) or melatonin (100 microM) to the medium during kainate or ischaemia treatments largely prevented the detrimental influences on the serotonin-accumulating and GABA cells. It is concluded that during experimental ischaemia excessive glutamate is released to influence cells which contain kainate and APB-type receptors. However, only the neurones containing the kainate receptors are negatively affected with the generation of free radicals. Melatonin or CNQX protects against this effect by scavenging free radicals or acting at the receptor level, respectively.

In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent.

The aim of the study was to determine whether betaxolol is a neuroprotective agent and can therefore slow down the changes seen in the retina following ischaemia/reperfusion. Ischaemia was induced in one rat eye by raising the intraocular pressure for 45 min. Three days later electroretinograms were recorded from both eyes and the retinas were examined immunohistochemically for the localisation of calretinin and choline acetyltransferase (ChAT) immunoreactivities. The effect of glutamate agonists, hypoxia or experimental ischaemia was examined on the GABA immunoreactivity, lactate dehydrogenase (LDH) and internal calcium levels ([Ca2+]i) of the isolated rabbit retina, rat cortical cultures and chick retinal cell cultures respectively. Betaxolol was tested to see whether it can attenuate the influence of the glutamate agonists, hypoxia or experimental ischaemia. Ischaemia for 45 min causes a change in the nature of the normal calretinin immunoreactivity, an obliteration of the ChAT immunoreactivity and a drastic reduction in the b-wave of the electroretinogram after 3 days of reperfusion. When betaxolol was injected i.p. into the rats before ischaemia and on the days of reperfusion the changes to the calretinin and ChAT immunoreactivities were reduced and the reduction of the b-wave was prevented. Rabbit retinas incubated in vitro in physiological solution lacking oxygen/glucose or containing the glutamate agonists kainate or NMDA caused a change in the nature of the GABA immunoreactivity. Inclusion of betaxolol partially prevented the changes caused by NMDA and lack of oxygen/glucose. Rat cortical cultures exposed to glutamate or hypoxia/reoxygenation resulted in a release of LDH. The release of the enzyme was almost completely attenuated when betaxolol was included in the culture medium. Kainate increased the [Ca2+]i in chick retinal cultures, as measured with Indo-1. In a medium with sodium, this kainate-induced elevation of [Ca2+]i was significantly reduced by betaxolol. The combined data show that betaxolol is a neuroprotective agent and attenuates the effects on the retina induced by raising the intraocular pressure to simulate an ischaemic insult as may occur in glaucoma.

Calcium-dependent free radical generation in cultured retinal neurons injured by kainate.

Cultured rat retinal neurons exposed to kainate produced free radicals, as demonstrated by electron spin resonance (ESR) spin trapping using the nitrone 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the generation of DMPO hydroxyl adduct (DMPO-OH). This DMPO-OH production was abolished by EGTA, nitro-arginine and oxypurinol, suggesting that it was dependent on Ca2+ influx and subsequent activation of nitric oxide synthase and xanthine oxidase. Moreover, kainate induced a receptor-mediated Ca2+ influx and neuronal injury assessed by lactate dehydrogenase release. Neuroprotection afforded by nitro-arginine and oxypurinol shows that calcium-dependent free radical production plays a major role in kainate retinal toxicity.

Protection by prostaglandins from glutamate toxicity in cortical neurons.

The growing evidence that glutamate may be an important agent mediating ischemic damage to neurons, led us to investigate the possible protective effects of pharmacological agents against glutamate in a model system of cortical neurons. In this study we examined, in particular, the cytoprotective effect of prostaglandins. Experiments were carried out in vitro by using rat cortical neurons in culture for 10 days. They were incubated for 3h with glutamate (10 microM) in the presence or absence of various pharmacological agents including prostaglandins (PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-Keto-PGF1 alpha, carba-TXA2, carba-PGI2 and PGF2 alpha-methylester). Increase in lacticodehydrogenase (LDH) release into the culture medium has been measured as an index of cell injury. When neurons were incubated with glutamate they released LDH due to NMDA-receptor activation since D-L-2-amino-5-phosphonovaleric acid, a specific receptor antagonist, protected the cells. The protective activity of oxypurinol, amflutizole, superoxide dismutase, NG nitro-L-arginine and quinacrine, also suggests that xanthine oxidase activation, the generation of superoxide radical, and nitrix oxide, as well as phospholipase A2 stimulation are responsible for neuron injury (i.e. LDH release). All the tested prostaglandins, except PGF2 alpha-methylester, afforded significant protection at concentrations between 0.1 and 10 microM. The order of potency of the prostanoids was: PGF2 alpha = PGE2 > Carba-TXA2 > PGE1 > PGD2 > PGI2 = Carba-PGI2 > 6-Keto-PGF1 alpha. Additional experiments showed that prostaglandins did not compete for the NMDA binding site and that they did not inhibit free radical-related membrane damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin (PGI2) protects rat cortical neurons in culture against hypoxia/reoxygenation and glutamate-induced injury.

Arachidonic acid and its metabolites are released in brain extracellular fluids as a result of ischemia and may participate in either damaging or protecting neural tissues. This study investigates the neuroprotective effect of prostacyclin (PGI2) on hypoxia (5 h)/reoxygenation (3 h) and on the excitotoxic neurotransmitter, glutamate (10 microM), in rat cortical neuron cultures. At microM concentrations, PGI2 inhibits lactate dehydrogenase release, a cell-injury marker. These results, showing a direct cytoprotective effect of PGI2 on brain cells, reinforce its beneficial properties on vessels and circulating cells in cerebral ischemia.

Superoxide and nitric oxide cooperation in hypoxia/reoxygenation-induced neuron injury.

Oxygen-derived free radicals are implicated in hypoxia- and reoxygenation-related brain injury. In addition, excitatory amino acid neurotransmitters seem to be involved in this neurotoxicity and could act through the L-arginine/nitric oxide (NO) synthase pathway. In the present study we have used rat forebrain neurons in culture submitted to hypoxia/reoxygenation to investigate the relative role of free radicals, glutamate, and nitric oxide in hypoxic neuronal injury. Hypoxia (5 h) followed by reoxygenation (0-24 h) induced cell damage assessed by lacticodehydrogenase release into culture medium. Superoxide dismutase (SOD, 500 U/mL), D-L-2-amino-5-phosphonovaleric acid (100 microM), a glutamate receptor antagonist, and NG-nitro-L-arginine (100 microM), an NO synthase inhibitor, protected the neurons. The effect of NG-nitro-L-arginine was reversed by adding L-arginine (10 mM) in the culture medium, and hemoglobin, which scavenges NO, also afforded protection. Hypoxia (5 h) provoked glutamate release from neurons, and this effect was inhibited by SOD. Exogenous glutamate (1-100 microM) induced lacticodehydrogenase release, and this effect was inhibited by glutamate antagonism, NO synthase inhibition, or superoxide radical scavenging. These data are consistent with the following sequence of events in hypoxia-related neurotoxicity: free radical formation, glutamate release, and activation of NO synthase leading to superoxide and NO cooperative toxicity.