Changes In Calcium Homeostasis Research Articles

Calcineurin Inhibition at the Clinical Phase of Prion Disease Reduces Neurodegeneration, Improves Behavioral Alterations and Increases Animal Survival

Prion diseases are fatal neurodegenerative disorders characterized by a long pre-symptomatic phase followed by rapid and progressive clinical phase. Although rare in humans, the unconventional infectious nature of the disease raises the potential for an epidemic. Unfortunately, no treatment is currently available. The hallmark event in prion diseases is the accumulation of a misfolded and infectious form of the prion protein (PrPSc). Previous reports have shown that PrPSc induces endoplasmic reticulum stress and changes in calcium homeostasis in the brain of affected individuals. In this study we show that the calcium-dependent phosphatase Calcineurin (CaN) is hyperactivated both in vitro and in vivo as a result of PrPSc formation. CaN activation mediates prion-induced neurodegeneration, suggesting that inhibition of this phosphatase could be a target for therapy. To test this hypothesis, prion infected wild type mice were treated intra-peritoneally with the CaN inhibitor FK506 at the clinical phase of the disease. Treated animals exhibited reduced severity of the clinical abnormalities and increased survival time compared to vehicle treated controls. Treatment also led to a significant increase in the brain levels of the CaN downstream targets pCREB and pBAD, which paralleled the decrease of CaN activity. Importantly, we observed a lower degree of neurodegeneration in animals treated with the drug as revealed by a higher number of neurons and a lower quantity of degenerating nerve cells. These changes were not dependent on PrPSc formation, since the protein accumulated in the brain to the same levels as in the untreated mice. Our findings contribute to an understanding of the mechanism of neurodegeneration in prion diseases and more importantly may provide a novel strategy for therapy that is beneficial at the clinical phase of the disease.

Isoproterenol effects on the contractility of papillary muscles in the heart of ground squirrel

This paper presents a study of the influence of isoproterenol (1 μM) on the force of isometric contractions (0.1–1.0 Hz; 30 ± 1°C; 1.8 mM Ca2+) of papillary muscles of the right ventricle in the heart of a ground squirrel during summer activity (n = 5) and hibernation season (activity between hibernation bouts, n = 4; torpor, n = 4; and arousal, n = 5). It is shown that isoproterenol increases the force of contraction (a positive inotropic effect) by 20 ± 3% and 61 ± 7% at stimulation frequencies of 0.4 and 1.0 Hz, respectively. In animals of hibernating period the isoproterenol-induced increase in the force of contraction is rather brief (within 3 min after onset of the influence) and is accompanied by a 30–50% decrease in the force from the control level (a negative inotropic effect) at stimulation frequencies from 0.3 to 0.8 Hz. The positive isoproterenol inotropic effect in active summer ground squirrels is associated with a decrease in a relative value of the pause potentiating effect (a qualitative indicator of calcium content in sarcoplasmic reticulum), and the negative inotropic effect, with its increase. In all groups of animals under examination the isoproterenol inotropic effect (regardless of its direction) is accompanied by the acceleration of the temporal parameters of the contraction—relaxation cycle. The dependence of isoproterenol effects in the heart of hibernating animals on both seasonal changes in calcium homeostasis and the activity of the sympathetic nervous system is under discussion.

Immunohistochemical localization of calbindin D28-k, parvalbumin, and calretinin in the cerebellar cortex of the circling mouse

The spontaneous mutant circling mouse has an autosomal recessive pattern of inheritance and is an animal model for deafness, which is characterized by circling, head tossing, and hyperactivity. Since the main pathology in circling mice lies in the organ of Corti, most studies on deaf mice have focused on auditory brain stem nuclei. No studies regarding behavior-related CNS changes in circling mice have been reported. The major center of sensory input for modulation of motor activity is best-studied in the cerebellum. Considering the importance of calcium homeostasis in numerous processes, calcium-binding proteins (CaBPs), such as calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), may play crucial roles in preserving cerebellar coordinated motor function. Thus, the distribution of CB, PV, and CR was determined in the cerebellum using immunohistochemical methods to compare immunoreactivity (IR) of CaBPs between wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice. The IR of CB and PV was predominantly observed in the Purkinje cell layer of all three genotypes. Compared with the +/+ genotype, the relative mean density of CB and PV IR in the Purkinje cell layer and CR IR in the granular layer was significantly decreased in the cir/cir genotype. Changes in calcium homeostasis in parallel fiber/Purkinje cell synapses could diminish cerebellar control of motor coordination. A number of deficiencies among the CaBPs lead to distinct alterations in brain physiology, which may affect normal behavior.

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca(2+)](i)) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH(4)Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at -80mV, NH(4)Cl triggered an inward current, the main component of which is a Ca(2+)-dependent chloride current. Calcium imaging confirmed that NH(4)Cl provoked a [Ca(2+)](i) increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I(In)). These currents were stimulated during external addition of NH(4)Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH(4)Cl and extracellular acidic medium, was shown to trigger a [Ca(2+)](i) increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH(4)Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.

A Mitocentric View of Alzheimer's Disease Suggests Multi-Faceted Treatments

Alzheimer's disease (AD) is defined by senile plaques made of amyloid-beta peptide (Abeta), neurofibrillary tangles made of hyperphosphorylated tau proteins, and memory deficits. Thus, the events initiating the cascade leading to these end points may be more effective therapeutic targets than treating each facet individually. In the small percentage of cases of AD that are genetic (or animal models that reflect this form of AD), the factor initiating AD is clear (e.g., genetic mutations lead to high Abeta1-42 or hyperphosphorylated tau proteins). In the vast majority of AD cases, the cause is unknown. Substantial evidence now suggests that abnormalities in glucose metabolism/mitochondrial function/oxidative stress (GMO) are an invariant feature of AD and occur at an early stage of the disease process in both genetic and non-genetic forms of AD. Indeed, decreases in brain glucose utilization are diagnostic for AD. Changes in calcium homeostasis also precede clinical manifestations of AD. Abnormal GMO can lead to plaques, tangles, and the calcium abnormalities that accompany AD. Abnormalities in GMO diminish the ability of the brain to adapt. Therapies targeting mitochondria may ameliorate abnormalities in plaques, tangles, calcium homeostasis, and cognition that comprise AD.

The relationship between cellular and calcium responses of Aspergillus awamori to external influences

The cellular and Ca2+ responses to physiological stimuli of different nature were studied in the experiments with the strain Aspergillus awamori 66A containing recombinant aequorin, a Ca2+-dependent photosensitive protein. The relationship between the cellular response registered by changes in the development of the mycelial fungus (colony growth, hyphal branching, and the rate of spore formation) and the level and duration of calcium flares in the cytosol was assessed. The physical or chemical stimuli (mechanical effect, osmotic shock) inducing short-time calcium flares in the cytosol did not influence significantly the development of A. awamori grown in liquid or on solid nutrient media. The action on the 24-h A. awamori culture of the Ca2+-selective ionophore (A23187) inducing long-term changes in calcium homeostasis caused disorders in the fungus development and morphology (hyperbranching of mycelial hyphae, formation of spherical cells, and inhibition of colony growth and spore formation). Thus, it was established that the development of cellular response in the micromycete correlated with the duration of the calcium flare in the cytosol.

Taurine regulates insulin release from pancreatic beta cell lines

BackgroundPancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.MethodsPancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.ResultsIn both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.ConclusionsTaurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

Cellular protective action of angiotensin converting enzyme inhibitors.

In several studies organ protective effects of ACE inhibitors independent from their antihypertensive action have been demonstrated. The mechanisms of the protective effects of angiotensin converting enzyme (ACE) inhibitors on vasculature and kidney are largely unknown. In the present study the modulatory action of captopril on the angiotensin II (AngII), arginine vasopressin (AVP), and platelet derived growth factor (PDGF)-induced increase of cytosolic free calcium concentration ([Ca2+]i) was investigated in cultured vascular smooth muscle cells (VSMC) and cultured glomerular mesangial cells (MC) from rats using the fluorescent dye technique. Resting [Ca2+]i in VSMC or MC was not significantly affected by captopril. The preincubation of VSMC with 1 mumol/l captopril significantly reduced the AngII-induced [Ca2+]i increase in VSMC from 90 +/- 10 nmol/l (n = 78; mean +/- SEM) to 51 +/- 16 nmol/l (n = 53; p < 0.05) and in MC from 102 +/- 42 nmol/l (n = 14) to 43 +/- 12 nmol/l (n = 7; p < 0.05). In the absence of extracellular calcium captopril produced no effect on AngII induced changes of [Ca2+]i. Captopril significantly attenuated the AVP-induced [Ca2+]i increase in VSMC and MC. The preincubation of MC with 1 mumol/l captopril for 40 min significantly reduced the PDGF-induced [Ca2+]i increase in MC from 127 +/- 31 nmol/l (n = 11) to 61 +/- 32 nmol/l (n = 5, p < 0.05). The present results may indicate that part of the protective effects of ACE inhibitors on vasculature and kidney may be promoted by inhibition of growth-factor induced changes of calcium homeostasis.

Immunoreactivity of Calcium-Binding Proteins in the Central Auditory Nervous System of Aged Rats

While many factors contribute to aging, changes in calcium homeostasis and calcium related neuronal processes are likely to be important. High intracellular calcium is toxic to cells and alterations in calcium homeostasis are associated with changes in calcium-binding proteins, which confine free Ca(2+). We therefore assayed the expression of the calcium binding proteins calretinin and calbindin in the central auditory nervous system of rats. Using antibodies to calretinin and calbindin, we assayed their expression in the cochlear nucleus, superior olivary nucleus, inferior colliculus, medial geniculate body and auditory cortex of young (4 months old) and aged (24 months old) rats. Calretinin and calbindin staining intensity in neurons of the cochlear nucleus was significantly higher in aged than in young rats (p<0.05) The number and staining intensity of calretinin-positive neurons in the inferior colliculus, and of calbindin-positive neurons in the superior olivary nucleus were greater in aged than in young rats (p<0.05). These results suggest that auditory processing is altered during aging, which may be due to increased intracellular Ca(2+) concentration, consequently leading to increased immunoreactivity toward calcium-binding proteins.

Preliminary biological evaluation of poli(amidoamine) (PAMAM) dendrimer G3.5 on selected parameters of rat liver mitochondria

Polyamidoamino (PAMAM) dendrimer of generation 3.5 (G3.5) specific interactions with rat liver mitochondria were studied. Selected parameters of mitochondria (transmembrane potential and uptake of Ca 2+ ions) were investigated after the exposure to G3.5 used at the concentration of 10, 30 and 50 μM and Ca 2+ ions used at 1 mM. The times of preincubation of isolated liver mitochondria with dendrimer were 5, 15 and 30 min at room temperature. The mitochondrial membrane potential was monitored spectrofluorimetrically by fluorescence quenching of Rhodamine 123 (Rh 123). The changes in calcium homeostasis upon dendrimer exposure were detected using flow cytometric analysis with Fluo-3. On the one hand the obtained results revealed an impact of the tested chemical on rat liver mitochondrial function. We found that dendrimer G3.5 in a concentration above 10 μM contributes to the reduction in the transmembrane potential and hinders in the influx of Ca 2+ ions to mitochondria added externally, or accelerates their efflux from mitochondria. The most effective preincubation time was observed to be 15 min for all tested concentrations. On the other hand the combined treatment of dendrimer G3.5 with Ca 2+ demonstrated the protective effect of G3.5 against mitochondrial depolarization caused by calcium ions. These preliminary studies suggest that tested dendrimer can significantly affect the mitochondria and modulate their functionality.

Saline Infusion Through the Pancreatic Duct Leads to Changes in Calcium Homeostasis Similar to Those Observed in Acute Pancreatitis

This work focuses on studying the early events associated with pancreatic damage after retrograde infusion through the pancreatic duct in rats. We have analyzed changes in calcium homeostasis and secretory response in pancreatic acini from rats with taurocholate-induced acute pancreatitis. Moreover, in order to test whether pancreatic duct manipulation can trigger damage inside pancreatic acinar cells, we have studied both parameters in acini from animals infused with saline. Our study demonstrates that taurocholate causes evident damage to acinar cells, impairing both calcium homeostasis and secretory response to CCK. In saline, a significant decrease in calcium cytosolic response to CCK was observed. Calcium disturbances similar to those observed in acute pancreatitis appear before secretion blockade and inflammation processes in saline treated rats. These results could be interesting since pancreatitis is associated to clinical procedures that require duct manipulation such as endoscopic retrograde cholangiopancreatography.

Effect of hindlimb unloading on resting intracellular calcium in intrafusal fibers and ramp-and-hold stretches evoked responsiveness of soleus muscle spindles in conscious rats

The present study was to investigate and evaluate the dynamic changes of calcium homeostasis of soleus muscle spindle for the exploration of the potential mechanisms of muscle spindle degeneration induced by hindlimb unloading. We systematically observed the changes in immunoreactivity of calbindin D28K (CaBP-D28K), intracellular resting calcium in intrafusal fibers of soleus muscle spindle, and the responsiveness of muscle spindles to ramp-and-hold stretches after short- and long-term (3, 7, 14 d) hindlimb unloading. The immunoreactivity of CaBP-D28K started to decrease after 7-d hindlimb unloading, while its decrease was obviously different compared with the control group 14 d following the hindlimb unloading. The resting calcium concentration was increased significantly at 3 d, and reached the peak level 14 d after the hindlimb unloading. The responsiveness of muscle spindles, assessed by investigating Index3-L(2), Index3-L(3), and Index5, to ramp-and-hold stretches started to decrease during the period of 7-d hindlimb unloading. All Indexs, in particular Index3-L(3) and Index5, were significantly decreased at 14 d after the hindlimb unloading. The data suggest the disturbance of calcium homeostasis in intrafusal fibers during the exposure to hindlimb unloading might gradually influence the structure and function of muscle spindles.

PTH and 1.25 vitamin D response to a low-calcium diet is associated with bone mineral density in renal stone formers

Renal calcium stones and hypercalciuria are associated with a reduced bone mineral density (BMD). Therefore, the effect of changes in calcium homeostasis is of interest for both stones and bones. We hypothesized that the response of calciuria, parathyroid hormone (PTH) and 1.25 vitamin D to changes in dietary calcium might be related to BMD. A single-centre prospective interventional study of 94 hyper- and non-hypercalciuric calcium stone formers consecutively retrieved from our stone clinic. The patients were investigated on a free-choice diet, a low-calcium diet, while fasting and after an oral calcium load. Patient groups were defined according to lumbar BMD (z-score) obtained by dual X-ray absorptiometry (group 1: z-score <-0.5, n = 30; group 2: z-score -0.5-0.5, n = 36; group 3: z-score >0.5, n = 28). The effect of the dietary interventions on calciuria, 1.25 vitamin D and PTH in relation to BMD was measured. An inverse relationship between BMD and calciuria was observed on all four calcium intakes (P = 0.009). On a free-choice diet, 1.25 vitamin D and PTH levels were identical in the three patient groups. However, the relative responses of 1.25 vitamin D and PTH to the low-calcium diet were opposite in the three groups with the highest increase of 1.25 vitamin D in group 1 and the lowest in group 3, whereas PTH increase was most pronounced in group 3 and least in group 1. Calcium stone formers with a low lumbar BMD exhibit a blunted response of PTH release and an apparently overshooting production of 1.25 vitamin D following a low-calcium diet.

Calcium‐dependent protein kinase C alpha and the frequency‐dependent increase in phosphorylation of troponin I in failing hearts

In healthy myocardium the force‐frequency relation, one of the key factors modulating cardiac performance, is positive, while in failing myocardium a negative relation is found. This difference has been ascribed to changes in calcium homeostasis and, more recently, to changes in calcium sensitivity through increased phosphorylation of troponin I (TnI). Our objective was to determine whether altered activation of calcium‐dependent protein kinase C alpha (PKCα) determines the increase in phosphorylation of TnI with frequency in failing hearts.Right ventricular heart failure was induced by monocrotaline (MCT; 80mg/kg). After 4 weeks isovolumic pressure–frequency relations were determined in Langendorff‐perfused hearts in a control (n=19) and MCT‐treated group (n=19). Thereafter, hearts were kept quiescent or paced at 9 Hz for 60 min and flash frozen to determine phosphorylation of TnI and PKCα by 1D‐gel electrophoresis.The pressure–frequency relation was constant in control and negative in MCT group. Phosphorylation of TnI did not depend on frequency in control but increased in MCT group. Protein expression and phosphorylation levels of PKCα were both increased with frequency in MCT group.These results indicate that frequency‐dependent changes in PKC‐phosphorylatable sites of TnI in failing hearts originate from an altered balance in calcium‐dependent kinase/phosphatase activity.

β-amyloid-induced changes in calcium homeostasis in cultured hippocampal neurons of the rat

A two-wave technique of calciometry with the use of a fluorescence dye, fura-2/AM, was applied for examination of the effect of a protein, β-amyloid (the main component of senile plaques in Alzheimer’s disease), on calcium homeostasis in cultured neurons of the rat hippocampus; β-amyloid was added to the culture medium. In most neurons, the effect of β-amyloid appeared as a more than twofold increase in the basic calcium concentration, as compared with the control (153.4 ± 11.5 and 71.7 ± 5.4 nM, respectively; P < 0.05). The characteristics of calcium transients induced by application of hyperpotassium solution also changed; the amplitude of these transients decreased, and the duration of a part corresponding to calcium release from the cell (rundown of the transient) increased. The mean amplitude of calcium transients under control conditions was 447.5 ± 20.1 nM, while after incubation in the presence of β-amyloid this index dropped to 278.4 ± 22.6 nM. Under control conditions, the decline phase of calcium transients lasted, on average, 100 ± 6 sec, while after incubation of hippocampal cell cultures in the presence of β-amyloid this phase lasted 250 ± 10 sec. Therefore, an excess of β-amyloid influences significantly calcium homeostasis in the nerve cells by disturbing functions of the calcium-controlling systems, such as voltage-operated calcium channels of the plasma membrane and calcium stores of the mitochondria and endoplasmic reticulum.

Linking of Autophagy to Ubiquitin-Proteasome System Is Important for the Regulation of Endoplasmic Reticulum Stress and Cell Viability

Two major protein degradation systems exist in cells, the ubiquitin proteasome system and the autophagy machinery. Here, we investigated the functional relationship of the two systems and the underlying mechanisms. Proteasome inhibition activated autophagy, suggesting that the two are functionally coupled. Autophagy played a compensatory role as suppression of autophagy promoted the accumulation of polyubiquitinated protein aggregates. Autophagy was likely activated in response to endoplasmic reticulum stress caused by misfolded proteins during proteasome inhibition. Suppression of a major unfolded protein response pathway mediated by IRE1 by either gene deletion or RNA interference dramatically suppressed the activation of autophagy by proteasome inhibitors. Interestingly, c-Jun NH(2)-terminal kinase (JNK) but not XBP-1, both of which are the known downstream targets of IRE1, seemed to participate in autophagy induction by proteasome inhibitors. Finally, proteasome inhibitor-induced autophagy was important for controlling endoplasmic reticulum stress and reducing cell death in cancer cells. Our studies thus provide a mechanistic view and elucidate the functional significance of the link between the two protein degradation systems.

Central core disease

Central core disease (CCD) is an inherited neuromuscular disorder characterised by central cores on muscle biopsy and clinical features of a congenital myopathy. Prevalence is unknown but the condition is probably more common than other congenital myopathies. CCD typically presents in infancy with hypotonia and motor developmental delay and is characterized by predominantly proximal weakness pronounced in the hip girdle; orthopaedic complications are common and malignant hyperthermia susceptibility (MHS) is a frequent complication. CCD and MHS are allelic conditions both due to (predominantly dominant) mutations in the skeletal muscle ryanodine receptor (RYR1) gene, encoding the principal skeletal muscle sarcoplasmic reticulum calcium release channel (RyR1). Altered excitability and/or changes in calcium homeostasis within muscle cells due to mutation-induced conformational changes of the RyR protein are considered the main pathogenetic mechanism(s). The diagnosis of CCD is based on the presence of suggestive clinical features and central cores on muscle biopsy; muscle MRI may show a characteristic pattern of selective muscle involvement and aid the diagnosis in cases with equivocal histopathological findings. Mutational analysis of the RYR1 gene may provide genetic confirmation of the diagnosis. Management is mainly supportive and has to anticipate susceptibility to potentially life-threatening reactions to general anaesthesia. Further evaluation of the underlying molecular mechanisms may provide the basis for future rational pharmacological treatment. In the majority of patients, weakness is static or only slowly progressive, with a favourable long-term outcome.

Physiological roles of muscle-derived interleukin-6 in response to exercise

To discuss recent findings with regard to the regulation of muscle-derived interleukin-6 as well as the possible physiological and metabolic roles of interleukin-6 in response to exercise. Contraction-induced transcription and release of interleukin-6 is primarily regulated by an altered intramuscular milieu in response to exercise. Accordingly, changes in calcium homeostasis, impaired glucose availability and increased formation of reactive oxygen species are all associated with exercise and capable of activating transcription factors known to regulate interleukin-6 synthesis. Acute interleukin-6 administration to humans increases lipolysis, fat oxidation and insulin-mediated glucose disposal. Adenosine monophosphate-activated protein kinase activation by interleukin-6 appears to play an important role in modulating some of these metabolic effects. Interleukin-6 facilitates an antiinflammatory milieu and may exert some of its biological effects via inhibition of the proinflammatory cytokine tumor necrosis factor-alpha. The discovery of contracting muscle as a cytokine-producing organ opens a new paradigm: skeletal muscle is an endocrine organ that in response to contractions produces and releases 'myokines', which subsequently can modulate the metabolic and immunological response to exercise in several tissues. In our view, interleukin-6 may be one of several myokines.

Lithium toxicity and expression of stress-related genes or proteins in A549 cells

To unveil some molecular mechanisms underlying lithium toxicity, expression changes of stress-related genes or proteins were analysed in A549 cells, cultured for 3 days in presence of lithium. A dose-dependant cell-growth inhibition was found for concentrations ranging from 2 (toxicity threshold) to 12 mM (lethality threshold). cDNA arrays technology was used to analyse effects of 5 and 10 mM lithium. Among genes involved in cell cycle regulation, proliferating cell nuclear antigen (PCNA) was down-regulated and cyclin kinase inhibitor p21 (CDKN1A), up-regulated. Genes of paraoxonase 2, known to prevent LDL lipid peroxidation, and of catalase and SOD were found to be down-regulated whereas genes of cytochrome P450 (CYP2F1, CYP2E1) were up-regulated. This probably results in higher intracellular levels of reactive oxygen species and account for increased levels of lipid peroxidation commonly associated with lithium exposure. Moreover, lithium was found to down-regulate genes coding for anti-apoptotic gene BAG-1 and for most of the molecular chaperones (HSP, GRP). This might account for lithium toxicity since these proteins are critical for cell survival. At translational level, a 105 kDa protein was found to be over-expressed. This protein was recognized by the anti-GRP94, anti-KDEL and anti-phosphoserine monoclonal antibodies suggesting that, lithium could induce post-translational modifications of GRP94 phosphorylation. Using tunicamycin and thapsigargin, it was concluded that lithium effects are not related to defect in N-linked glycosylation and/or to changes in calcium homeostasis.

Toxicity of β-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl- d-aspartate receptor subunits

Neurotoxicity induced by beta-amyloid peptide (Aβ) involves glutamate toxicity, resulting from overactivation of N-methyl- d-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Aβ 1–40 toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Aβ 1–40 toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Aβ 1–40 decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Aβ 1–40. Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Aβ 1–40 through changes in calcium homeostasis.