Calcium reabsorption in the distal tubule: regulation by sodium, pH, and flow.

We developed a mathematical model of Ca transport along the late distal convoluted tubule (DCT2) and the connecting tubule (CNT) to investigate the mechanisms that regulate Ca reabsorption in the DCT2-CNT. The model accounts for apical Ca influx across transient receptor potential vanilloid 5 (TRPV5) channels and basolateral Ca efflux via plasma membrane Ca-ATPase pumps and type 1 Na/Ca exchangers (NCX1). Model simulations reproduce experimentally observed variations in Ca uptake as a function of extracellular pH, Na, and Mg concentration. Our results indicate that amiloride enhances Ca reabsorption in the DCT2-CNT predominantly by increasing the driving force across NCX1, thereby stimulating Ca efflux. They also suggest that because aldosterone upregulates both apical and basolateral Na transport pathways, it has a lesser impact on Ca reabsorption than amiloride. Conversely, the model predicts that full NCX1 inhibition and parathyroidectomy each augment the Ca load delivered to the collecting duct severalfold. In addition, our results suggest that regulation of TRPV5 activity by luminal pH has a small impact, per se, on transepithelial Ca fluxes; the reduction in Ca reabsorption induced by metabolic acidosis likely stems from decreases in TRPV5 expression. In contrast, elevations in luminal Ca are predicted to significantly decrease TRPV5 activity via the Ca-sensing receptor. Nevertheless, following the administration of furosemide, the calcium-sensing receptor-mediated increase in Ca reabsorption in the DCT2-CNT is calculated to be insufficient to prevent hypercalciuria. Altogether, our model predicts complex interactions between calcium and sodium reabsorption in the DCT2-CNT.

Calcium Microdomains Control Exo-Endocytosis of Synaptic-Like Microvesicles in Astrocytes

During the last decade, the discovery that astrocytes possess a nonelectrical form of excitability (Ca21-excitability) that leads to the release of chemical transmitters, an activity called ''gliotransmission'', indicates that these cells may have additional important roles in brain function. Elucidating the stimulus-secretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. Here by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we investigated the spatiotemporal characteristics of stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes. We performed the analysis at both the whole-cell and single-vesicle levels providing the first system for comparing exo-endocytic processes in astrocytes with those in neurons. Both the time course and modalities of secretion in astrocytes present more similarities to neurons then previously expected. We found that 1. the G-protein-coupled receptor (GPCR)-evoked exocytosis reached the maximum on a ms time scale and that 2. ER tubuli formed sub-micrometer domains beneath the plasma membrane in close proximity to exocytic vesicles, where fusion events were spatiotemporally correlated with fast Ca21 events.

Role of homer 1 proteins in the calcium signaling pathway(s) underlying exo-endocytosis processes of glutamatergic slmvs in astrocytes

The discovery that astrocytes possess a nonelectrical form of excitability (calcium excitability) that leads to the release of chemical transmitters, an activity called gliotransmission, indicates that these cells may have additional important roles in brain function. Elucidating the stimulussecretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. We have recently discovered the existence in astrocytes of functional sub-membrane microdomains of calcium release from the internal stores in response to mGluR5 activation (Marchaland et al., J of Neurosci., 2008). Such sub-plasma membrane calcium microdomains control exocytosis of astrocytic glutamate signaling to neurons. Homer proteins are scaffold proteins controlling calcium signaling in different cellular microdomains, including dendritic spines in neurons (Sala et al., J of Neurosci., 2005). Thus, similarly to dendritic pines, Homer1 could be implicated in the coupling between astrocytic mGluR5 and IP3Rs on the ER. Here, by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we investigated the involvement of Homer1 proteins in the calcium dependent stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes.

Dairy calcium supplementation in overweight or obese persons: its effect on markers of fat metabolism

BACKGROUND: Dairy calcium supplementation has been proposed to increase fat oxidation and to inhibit lipogenesis. OBJECTIVE: We aimed to investigate the effects of calcium supplementation on markers of fat metabolism. DESIGN: In a placebo-controlled, crossover experiment, 10 overweight or obese subjects who were low calcium consumers received 800 mg dairy Ca/d for 5 wk. After 4 wk, adipose tissue was taken for biopsy for analysis of gene expression. Respiratory exchange, glycerol turnover, and subcutaneous adipose tissue microdialysis were performed for 7 h after consumption of 400 mg Ca or placebo, and the ingestion of either randomized slow-release caffeine (SRC; 300 mg) or lactose (500 mg). One week later, the test was repeated with the SRC or lactose crossover. RESULTS: Calcium supplementation increased urinary calcium excretion by 16% (P = 0.017) but did not alter plasma parathyroid hormone or osteocalcin concentrations. Resting energy expenditure (59.9 +/- 3.0 or 59.6 +/- 3.3 kcal/h), fat oxidation (58.4 +/- 2.5 or 53.8 +/- 2.2 mg/min), plasma free fatty acid concentrations (0.63 +/- 0.02 or 0.62 +/- 0.03 mmol/L), and glycerol turnover (3.63 +/- 0.41 or 3.70 +/- 0.38 micromol . kg(-1) . min(-1)) were similar with or without calcium, respectively. SRC significantly increased free fatty acid concentrations, resting fat oxidation, and resting energy expenditure. During microdialysis, epinephrine increased dialysate glycerol concentrations by 250% without and 254% with calcium. Expression of 7 key metabolic genes in subcutaneous adipose tissue was not affected by calcium supplementation. CONCLUSION: Dairy calcium supplementation in overweight subjects with habitually low calcium intakes failed to alter fat metabolism and energy expenditure under resting conditions and during acute stimulation by caffeine or epinephrine

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Multi-analyte detection for biological fluids. Towards continous monitoring of glucose, ionized calcium and pH using a viscometric affinity biosensor.

We present a viscometric affinity biosensor that can potentially allow continuous multi-analyte monitoring in biological fluids like blood or plasma. The sensing principle is based on the detection of viscosity changes of a polymeric solution which has a selective affinity for the analyte of interest. The chemico-mechanical sensor incorporates an actuating piezoelectric diaphragm, a sensing piezoelectric diaphragm and a flow-resisting microchannel for viscosity detection. A free-standing Anodic Alumina Oxide (AAO) porous nano-membrane is used as selective interface. A glucose-sensitive sensor was fabricated and extensively assessed in buffer solution. The sensor reversibility, stability and sensitivity were excellent during at least 65 hours. Results showed also a good degree of stability for a long term measurement (25 days). The sensor behaviour was furthermore tested in fetal bovine serum (FBS). The obtained results for glucose sensing are very promising, indicating that the developed sensor is a candidate for continuous monitoring in biological fluids. Sensitive solutions for ionized calcium and pH are currently under development and should allow multi-analyte sensing in the near future.

Furosemide stimulation of parathormone in humans: role of the calcium-sensing receptor and the renin-angiotensin system.

Interactions between sodium and calcium regulating systems are poorly characterized but clinically important. Parathyroid hormone (PTH) levels are increased shortly after furosemide treatment by an unknown mechanism, and this effect is blunted by the previous administration of a calcimimetic in animal studies. Here, we explored further the possible underlying mechanisms of this observation in a randomized crossover placebo-controlled study performed in 18 human males. Volunteers took either cinacalcet (60 mg) or placebo and received a 20 mg furosemide injection 3 h later. Plasma samples were collected at 15-min intervals and analyzed for intact PTH, calcium, sodium, potassium, magnesium, phosphate, plasma renin activity (PRA), and aldosterone up to 6 h after furosemide injection. Urinary electrolyte excretion was also monitored. Subjects under placebo presented a sharp increase in PTH levels after furosemide injection. In the presence of cinacalcet, PTH levels were suppressed and marginal increase of PTH was observed. No significant changes in electrolytes and urinary excretion were identified that could explain the furosemide-induced increase in PTH levels. PRA and aldosterone were stimulated by furosemide injection but were not affected by previous cinacalcet ingestion. Expression of NKCC1, but not NKCC2, was found in parathyroid tissue. In conclusion, our results indicate that furosemide acutely stimulates PTH secretion in the absence of any detectable electrolyte changes in healthy adults. A possible direct effect of furosemide on parathyroid gland needs further studies.

Dietary calcium restriction enhances cadmium-induced metallothionein synthesis in rats.

Experiments were conducted with adult male rats to investigate the effects of dietary calcium (Ca) restriction upon intake and tissue distribution of cadmium (Cd), and Cd-metallothionein (Mt) synthesis. Four groups of animals were fed either a low-Ca, semisynthetic diet (0.1% Ca) or the same diet supplemented with 0.8% Ca (normal diet). The caloric intake was similar in all groups. Two groups (low-Ca and normal diet) were used as controls, and two groups (low-Ca and normal diet) received 100 mg/l Cd (as CdCl2) in drinking water. Cd levels in liver, kidney, spleen and red cells were measured in all animals after 8 weeks of treatment. Concomitantly, Mt levels in plasma, liver and kidney were evaluated by radioimmunoassay. Ca deficiency entailed marked and significant increases in accumulation of Cd and synthesis of Mt in all assayed tissues. It is concluded that dietary Ca restriction, independent of caloric intake, enhances Cd intestinal absorption and tissue accumulation, which is followed by increased tissue Mt synthesis.

Furosemide stimulation of parathormone in humans: role of the calcium-sensing receptor and the renin-angiotensin system

Les interactions entre les systèmes de régulation du sodium et du calcium sont encore mal comprises et leur importance clinique mérite d'être étudiée plus en détail. Les études chez l'animal ont montré qu'il existe des relations entre le taux plasmatique d'hormone parathyroïdienne (PTH) et l'aldostérone ou l'activité de la rénine. Par ailleurs, il a été démontré chez l'animal et chez l'homme que le taux sanguin de PTH augmente rapidement après une injection de furosémide, un diurétique de l'anse ce qui fait penser qu'il existe un lien entre l'effet du furosémide sur le rein et la sécrétion de PTH. Toutefois, à ce jour, le(s) mécanisme(s) impliqués dans ce lien reste(nt) encore inconnu(s). Des résultats plus récents suggèrent que l'effet du furosemide est amoindri par l'administration préalable d'un calcimimétique agissant au niveau du récepteur sensible au calcium (calcium sensing receptor). Pour explorer chez l'homme, les mécanismes possibles du lien entre PTH et effet du furosemide sur le rein, nous avons planifié une étude randomisée croisée contre placebo réalisée chez 18 volontaires sains masculins. Le but principal était d'investiguer le rôle du système rénine-angiotensine et des calcium sensing receptors. L'étude s'est donc réalisée en 2 phases pour chaque sujet. Les participants ont ainsi reçu soit du cinacalcet (60mg) soit un placebo dans une première phase et le placebo ou du cinacalcet dans la 2° phase. Dans chaque phase d'évaluation, une injection de 20 mg de furosemide a été administrée par voie intraveineuse à l'équilibre soit 3 heures après la prise du placebo ou du cinacalcet. Des échantillons de plasma ont été prélevés toutes les 15 minutes pendant 1 heure puis toutes les heures pour le dosage de PTH intacte, calcium, sodium, potassium, magnésium, phosphate, activité de la rénine plasmatique et aldostérone jusqu'à 6h après l'injection de furosémide. L'excrétion urinaire de ces mêmes électrolytes a été mesurée aux mêmes intervalles.

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10% calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49% of the platelets were classified as state of activation uncertain, 41% as resting, 9% as fully activated and 1% as irreversibly damaged. Treatment with 10% CaCl2 provided a distribution of 54% platelets in state of activation uncertain, 24% as fully activated, 20% as resting, and 2% as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10% CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets.

Coronary artery plaque burden and calcium scores in healthy men adhering to long-term wine drinking or alcohol abstinence

Observational studies suggest there are clinical benefits to moderate red wine (RW) consumption. However, the effects on coronary vasculature and overall lifestyle are unclear. We investigated whether a lifestyle of regular long-term RW consumption is associated with changes in coronary plaque burden, calcium score, carotid intima/media thickness, endothelial function, and metabolic variables, compared with alcohol abstinence. Healthy volunteers were evaluated by coronary computed tomography angiography (CTA) as well as carotid and brachial artery ultrasound. Nutritional status, psychological status, and metabolic variables were assessed. The study included 101 drinkers [aged 58.9±7.3 years (means±SD)], from wine brotherhoods, and 104 abstainers, from Anglican, Evangelical and Catholic churches both in the city of São Paulo, Brazil. No significant differences in demographics were noted. Lesion prevalence per patient assessed by coronary CTA and classified as absent (0), 1-25, 26-49, and ≥50% stenosis was similar between groups. When analyzed by individual arteries, i.e., left anterior descending, circumflex, and right coronary, prevalence was also not different. On the other hand, calcium scores were higher among drinkers than abstainers (144.4±362.2 vs 122.0±370.3; P<0.01). However, drinkers reported less history of diabetes and exercised more. RW drinkers consumed 2127.9±387.7 kcal/day while abstainers consumed 1836.0±305.0 (P<0.0001). HDL cholesterol was significantly higher among drinkers compared to abstainers (46.9±10.9 vs 39.5±9.0 mg/dL; P<0.001), while fasting plasma glucose was lower (97.6±18.2 vs 118.4±29.6 mg/dL; P<0.02). Liver enzymes were normal in both groups. In conclusion, long-term wine drinkers displayed a similar plaque burden but greater calcium score than abstainers, despite a more atherogenic diet, and the mechanisms for the increased calcium scores in the former remain speculative.

Investigation into the determination of arsenic by direct current plasma atomic emission spectrometer

Improvements have been made on the currently available hydride generator system manufactured by SpectraMetrics Incorporated, because the system was found to be unsatisfactory with respect to the following: 1. the drying agent, anhydrous calcium chloride, 2. the special sample tube, 3. the direction of argon flow through the Buchner funnel when it came to dealing with real sample, that is, with reference only to aqueous extracts of soil samples. Changes that were made on the system included the replacement of anhydrous calcium chloride with anhydrous calcium sulphate and the replacement of the special sample tube with a modified one made from silica. Re-directing the flow of argon through the top of the Buchner funnel appeared to make the system compatible with aqueous extracts of soil samples. The interferences from 1000 ~g/mL of nickel(II) , cobalt(II), iron(III), copper(II) have been eliminated with the aid of 1.4 M hydrochloric acid and 1% (weight/volume) L-cystine. Greater than 90% recovery of 0.3 ~g/mL arsenic signal was achieved in each case. Furthermore, 103% of arsenic signal was accomplished in the presence of 1000 ~g/mL cadmium with 5 M Hel. tVhen each of the interferents was present in solution at 1000 ppm, a recovery of 85% was achieved by using 5 M hydrochloric acid and 3% (weight/volume) L-cystine. Without L-cystine and when 1.4 M hydrochloric acid was used, the recoveries were 0% (Ni), 0% (Co), 88% (Fe), 15% (Cu), 18% (Cd). Similarly, a solution containing 1000 ppm of each interferent gave a zero percent recovery of arsenic. The reduction of trivalent and pentavalent arsenic at a pH less than one has also been investigated and shown to be quantitative if peak areas are measured. The reproducibility determination of a 0.3 Vg/mL standard arsenic solution by hydride generation shows a relative standard deviation of 3.4%. The detection limits with and without Porapak Q have been found to be 0.6 ng/mL and 1.0 ng/mL, respectively.

Effet du calcium, plomb et cuivre sur la bioaccumulation du cadmium et la production des phytochélatines par Chlamydomonas reinhardtii

Dans les milieux contaminés par les métaux, les organismes vivants sont exposés à plusieurs d’entre eux en même temps. Les modèles courants de prédiction des effets biologiques des métaux sur les organismes (p. ex., modèle du ligand biotique, BLM ; modèle de l’ion libre, FIAM), sont des modèles d’équilibre chimique qui prévoient, en présence d'un deuxième métal, une diminution de la bioaccumulation du métal d’intérêt et par la suite une atténuation de ses effets. Les biomarqueurs de toxicité, tels que les phytochélatines (PCs), ont été utilisés comme étant un moyen alternatif pour l’évaluation des effets biologiques. Les phytochélatines sont des polypeptides riches en cystéine dont la structure générale est (γ-glu-cys)n-Gly où n varie de 2 à 11. Leur synthèse semble dépendante de la concentration des ions métalliques ainsi que de la durée de l’ exposition de l’organisme, aux métaux. L'objectif de cette étude était donc de déterminer, dans les mélanges binaires de métaux, la possibilité de prédiction de la synthèse des phytochélatines par les modèles d’équilibres chimiques, tel que le BLM. Pour cela, la quantité de phytochélatines produites en réponse d’une exposition aux mélanges binaires : Cd-Ca, Cd-Cu et Cd-Pb a été mesurée tout en surveillant l’effet direct de la compétition par le biais des concentrations de métaux internalisés. En effet, après six heures d’exposition, la bioaccumulation de Cd diminue en présence du Ca et de très fortes concentrations de Pb et de Cu (de l’ordre de 5×10-6 M). Par contre, avec des concentrations modérées de ces deux métaux, le Cd augmente en présence de Cu et ne semble pas affecté par la présence de Pb. Dans le cas de la compétition Cd-Cu, une bonne corrélation a été observée entre la production de PC2, PC3 et PC4 et la quantité des métaux bioaccumulés. Pour la synthèse des phytochélatines et la bioaccumulation, les effets étaient considérés comme synergiques. Dans le cas du Cd-Ca, les quantités de PC3 et PC4 ont diminué avec le métal internalisé (effet antagoniste), mais ce qui était remarquable était la grande quantité de cystéine (GSH) et PC2 qui ont été produites à de fortes concentrations du Ca. Le Pb seul n’a pas induit les PCs. Par conséquent, il n’y avait pas de variation de la quantité de PCs avec la concentration de Pb à laquelle les algues ont été exposées. La détection et la quantification des PCs ont été faites par chromatographie à haute performance couplée d’un détecteur de fluorescence (HPLC-FL). Tandis que les concentrations métalliques intracellulaires ont été analysées par spectroscopie d’absorption atomique (AAS) ou par spectrométrie de masse à source plasma à couplage inductif (ICP-MS).

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS