Peculiarities of ion homeostasis in neurons containing calcium-permeable AMPA receptors.

Glutamate excitotoxicity accompanies numerous brain pathologies, including traumatic brain injury, ischemic stroke, and epilepsy. Disturbances of the ion homeostasis, mitochondria dysfunction, and further cell death are considered the main detrimental consequences of excitotoxicity. It is well known that neurons demonstrate different vulnerability to pathological exposures. In this regard, neurons containing calcium-permeable AMPA receptors (CP-AMPARs) may show higher susceptibility to excitotoxicity due to an additional pathway of Ca2+ influx. Here, we demonstrate that neurons containing CP-AMPARs are characterized by the higher amplitude of the glutamate-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and slower restoration of [Ca2+]i level compared to non-CP-AMPA neurons. Moreover, we have found that NASPM, an antagonist of CP-AMPARs, significantly decreases the amplitude of the [Ca2+]i elevation induced by glutamate or selective AMPARs agonist, 5-fluorowillardiine. In contrast, the antagonists of NMDARs or KARs affect insignificantly. We have also described some peculiarities of Na+, K+, and H+ intracellular dynamics in neurons containing CP-AMPARs. In particular, the amplitude of [Na+]i elevation was lower compared to non-CP-AMPA neurons, whereas the amplitude of [K+]i decrease was higher. We have shown the significant inverse correlation between [K+]i and [Ca2+]i and between intracellular pH and [Na+]i in CP-AMPARs-containing and non-CP-AMPA neurons upon glutamate excitotoxicity. Our data indicate that CP-AMPARs-mediated Ca2+ influx and slow removal of Ca2+ from the cytosol may underlie the vulnerability of the CP-AMPARs-containing neurons to glutamate excitotoxicity. Further studies of the mechanisms mediating the disturbances in ion homeostasis are crucial for developing new approaches for protecting these neurons at brain pathologies.

Of the Mechanisms of Paroxysmal Depolarization Shifts: Generation and Maintenance of Bicuculline-Induced Paroxysmal Activity in Rat Hippocampal Cell Cultures.

Abnormal depolarization of neuronal membranes called paroxysmal depolarization shift (PDS) represents a cellular correlate of interictal spikes. The mechanisms underlying the generation of PDSs or PDS clusters remain obscure. This study aimed to investigate the role of ionotropic glutamate receptors (iGluRs) in the generation of PDS and dependence of the PDS pattern on neuronal membrane potential. We have shown that significant depolarization or hyperpolarization (by more than ±50 mV) of a single neuron does not change the number of individual PDSs in the cluster, indicating the involvement of an external stimulus in PDS induction. Based on this data, we have suggested reliable protocols for stimulating single PDS or PDS clusters. Furthermore, we have found that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors are necessary for PDS generation since AMPAR antagonist NBQX completely suppresses bicuculline-induced paroxysmal activity. In turn, antagonists of NMDA (N-methyl-D-aspartate) and kainate receptors (D-AP5 and UBP310, respectively) caused a decrease in the amplitude of the first action potential in PDSs and in the amplitude of the oscillations of intracellular Ca2+ concentration occurring alongside the PDS cluster generation. The effects of the NMDAR (NMDA receptor) and KAR (kainate receptor) antagonists indicate that these receptors are involved only in the modulation of paroxysmal activity. We have also shown that agonists of some Gi-coupled receptors, such as A1 adenosine (A1Rs) or cannabinoid receptors (CBRs) (N6-cyclohexyladenosine and WIN 55,212-2, respectively), completely suppressed PDS generation, while the A1R agonist even prevented it. We hypothesized that the dynamics of extracellular glutamate concentration govern paroxysmal activity. Fine-tuning of neuronal activity via action on Gi-coupled receptors or iGluRs paves the way for the development of new approaches for epilepsy pharmacotherapy.

A novel approach for vital visualization and studying of neurons containing Ca2+ -permeable AMPA receptors.

Calcium-permeable AMPA receptors (CP-AMPARs) play a pivotal role in brain functioning in health and disease. They are involved in synaptic plasticity, synaptogenesis, and neuronal circuits development. However, the functions of neurons expressing CP-AMPARs and their role in the modulation of network activity remain elusive since reliable and accurate visualization methods are absent. Here we developed an approach allowing the vital identification of neurons containing CP-AMPARs. The proposed method relies on evaluating Ca2+ influx in neurons during activation of AMPARs in the presence of NMDAR and KAR antagonists, and blockers of voltage-gated Ca2+ channels. Using this method, we studied the properties of CP-AMPARs-containing neurons. We showed that the overwhelming majority of neurons containing CP-AMPARs are GABAergic, and they are distinguished by higher amplitudes of the calcium responses to applications of the agonists. Furthermore, about 30% of CP-AMPARs-containing neurons demonstrate the presence of GluK1-containing KARs. Although CP-AMPARs-containing neurons are characterized by more significant Ca2+ influx during the activation of AMPARs than other neurons, AMPAR-mediated Na+ influx is similar in these two groups. We revealed that neurons containing CP-AMPARs demonstrate weak GABA(A)R-mediated inhibition because of the low percentage of GABAergic synapses on the soma of these cells. However, our data show that weak GABA(A)R-mediated inhibition is inherent to all GABAergic neurons in the culture and cannot be considered a unique feature of CP-AMPARs-containing neurons. We believe that the suggested approach will help to understand the role of CP-AMPARs in the mammalian nervous system in more detail.

Molecular and Cellular Interactions in Pathogenesis of Sporadic Parkinson Disease.

An increasing number of the population all around the world suffer from age-associated neurodegenerative diseases including Parkinson's disease (PD). This disorder presents different signs of genetic, epigenetic and environmental origin, and molecular, cellular and intracellular dysfunction. At the molecular level, α-synuclein (αSyn) was identified as the principal molecule constituting the Lewy bodies (LB). The gut microbiota participates in the pathogenesis of PD and may contribute to the loss of dopaminergic neurons through mitochondrial dysfunction. The most important pathogenetic link is an imbalance of Ca2+ ions, which is associated with redox imbalance in the cells and increased generation of reactive oxygen species (ROS). In this review, genetic, epigenetic and environmental factors that cause these disorders and their cause-and-effect relationships are considered. As a constituent of environmental factors, the example of organophosphates (OPs) is also reviewed. The role of endothelial damage in the pathogenesis of PD is discussed, and a 'triple hit hypothesis' is proposed as a modification of Braak's dual hit one. In the absence of effective therapies for neurodegenerative diseases, more and more evidence is emerging about the positive impact of nutritional structure and healthy lifestyle on the state of blood vessels and the risk of developing these diseases.

Role of L-Type Voltage-Gated Calcium Channels in Epileptiform Activity of Neurons.

Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca2+ concentration ([Ca2+]i) in neurons. Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 µM demonstrate greater selectivity than the blockers from other groups (phenylalkylamines and benzothiazepines). However, high doses of dihydropyridines evoke an irreversible increase in [Ca2+]i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1-10 µM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca2+]i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca2+]i level during oscillations, which appears to determine the number of PDSs in the cluster.

Mechanisms of ammonium-induced neurotoxicity. Neuroprotective effect of alpha-2 adrenergic agonists.

Here we report the effects of ammonium on the main biophysical features of neurons and astrocytes during the first minutes of exposure. We found that ammonium causes the depolarization of neurons, which leads to the generation of high-frequency action potentials (APs). The initial alkalization and subsequent acidification of the intracellular medium in neurons occur along with the generation of calcium oscillations. Moreover, although the kinetics of calcium response of neurons and astrocytes is different, the dynamics of changes in the intracellular pH (pHi) is similar. The rate of superoxide production and mitochondrial membrane potential do not change in most neurons and astrocytes during ammonium exposure. At the same time, we observed an increased superoxide production and a decrease in the mitochondrial potential in some neurons in response to ammonium application. However, in both cases, the amplitude of the calcium response in these neurons is significantly higher compared to other neurons. Application of UK 14,304, an agonist of alpha-2 adrenergic receptors (A-2ARs), decreased the frequency of APs upon ammonium-induced high-frequency spike activity. Moreover, we also observed periods of hyperpolarization occurred in individual neurons. We suppose that this hyperpolarization contributes to the suppression of activity and can be mediated by astrocytic GABA release, which is stimulated upon activation of A-2ARs. Thus, our findings reveal a new possible mechanism of the protective action of alpha-2 adrenergic agonists against ammonium-induced hyperexcitation and demonstrate the correlation between intracellular calcium concentration, mitochondrial membrane potential, pHi, the intensity of superoxide production in hippocampal cells under acute hyperammonemia.

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes.

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH4 Cl-induced oscillations of the intracellular Ca2+ concentration ([Ca2+ ]i ) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca2+ ]i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca2+ ]i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca2+ ]i rise, mediated by Gißγ subunit and subsequent PLC-induced mobilization of Ca2+ from internal stores, and a plateau phase, mediated by a Ca2+ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca2+ response in astrocytes and suppression of [Ca2+ ]i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of Gßγ -signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca2+ ]i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via Gißγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.

Epileptiform activity promotes decreasing of Ca2+ conductivity of NMDARs, AMPARs, KARs, and voltage-gated calcium channels in Mg2+-free model.

NMDA, AMPA, and kainate receptors are the principal excitatory receptors in the brain. These receptors have been considered as the main targets in the treatment of epilepsy in recent years. This work aimed to determine how the Ca2+ conductivity of ionotropic glutamate receptors and voltage-gated Ca2+ channels changes in an in vitro model of epilepsy. For induction of epileptiform activity, hippocampal neurons were exposed to Mg2+-free medium. It has been shown that removal of Mg2+ from the medium not only removes the block from the NMDA receptors but also stimulates the release of glutamate in a way that is independent of the NMDA receptors. Under these conditions, the structure of the bursts significantly differs from the spontaneous bursts arising in mature hippocampal cultures. We have demonstrated that the frequency and amplitude of Mg2+-free medium-induced Ca2+ oscillations decrease after the 60-min exposure. Besides, the Ca2+ conductivity of ionotropic glutamate receptors and voltage-gated calcium channels significantly reduces. Thus, the decrease of Ca2+ conductivity can be considered as one of the mechanisms of adaptation during epilepsy.

Domoic acid suppresses hyperexcitation in the network due to activation of kainate receptors of GABAergic neurons.

Kainate receptors play an important role in the brain. They contribute to postsynaptic depolarization, modulate the release of neurotransmitters such as GABA and glutamate, affect the development of the neuronal network. At the same time, their functions depend not only on the type of neuron expressing them but also on their localization (pre- or postsynaptic). It has been shown in present work that activation of kainate receptors by domoic acid stimulates the secretion of both glutamate and GABA. This effect is observed at a concentration of 100 nM. At higher levels (200-500 nM), domoic acid selectively activates a specific population of GABAergic neurons. The peculiarity of these neurons is increased excitability in the network. This phenomenon can be explained by the weak GABA(A)R-mediated inhibition, as well as by the lower activation threshold of voltage-gated channels. Moreover, activation of these GABAergic neurons by domoic acid leads to the suppression of activity in the network under ammonium-induced hyperexcitation. As shown by inhibitory analysis, this effect is mediated by GABA(A) receptors. The obtained data may be of interest since the suppression of hyperexcitation via the selective activation of GABAergic neurons can be considered as a new potential approach to the treatment of diseases accompanied by increased neuronal activity such as epilepsy, ischemia and hepatic encephalopathy.

Role of DJ-1 in the mechanism of pathogenesis of Parkinson's disease.

DJ-1 protein has multiple specific mechanisms to protect dopaminergic neurons against neurodegeneration in Parkinson's disease. Wild type DJ-1 can acts as oxidative stress sensor and as an antioxidant. DJ-1 exhibits the properties of molecular chaperone, protease, glyoxalase, transcriptional regulator that protects mitochondria from oxidative stress. DJ-1 increases the expression of two mitochondrial uncoupling proteins (UCP 4 and UCP5), that decrease mitochondrial membrane potential and leads to the suppression of ROS production, optimizes of a number of mitochondrial functions, and is regarded as protection for the neuronal cell survival. We discuss also the stabilizing interaction of DJ-1 with the mitochondrial Bcl-xL protein, which regulates the activity of (Inositol trisphosphate receptor) IP3R, prevents the cytochrome c release from mitochondria and inhibits the apoptosis activation. Upon oxidative stress DJ-1 is able to regulate various transcription factors including nuclear factor Nrf2, PI3K/PKB, and p53 signal pathways. Stress-activated transcription factor Nrf2 regulates the pathways to protect cells against oxidative stress and metabolic pathways initiating the NADPH and ATP production. DJ-1 induces the Nrf2 dissociation from its inhibitor Keap1 (Kelch-like ECH-associated protein 1), promoting Nrf2 nuclear translocation and binding to antioxidant response elements. DJ-1 is shown to be a co-activator of the transcription factor NF-kB. Under nitrosative stress, DJ-1 may regulate PI3K/PKB signaling through PTEN transnitrosylation, which leads to inhibition of phosphatase activity. DJ-1 has a complex modulating effect on the p53 pathway: one side DJ-1 directly binds to p53 to restore its transcriptional activity and on the other hand DJ-1 can stimulate deacylation and suppress p53 transcriptional activity. The ability of the DJ-1 to induce activation of different transcriptional factors and change redox balance protect neurons against aggregation of α-synuclein and oligomer-induced neurodegeneration.

Attenuation of calmodulin regulation evokes Ca2+ oscillations: evidence for the involvement of intracellular arachidonate-activated channels and connexons.

Intracellular Са2+ controls its own level by regulation of Ca2+ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca2+ concentration in different cells. We have found persistent Са2+ oscillations in cultured white adipocytes in response to calmidazolium (CMZ). They appeared at [CMZ] > 1 µM as repetitive sharp spikes mainly superimposed on a transient or elevated baseline. Similar oscillations were observed when we used trifluoperazine. Oscillations evoked by 5 µM CMZ resulted from the release of stored Ca2+ and were supported by Са2+ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A2 inhibited by AACOCF3 was responsible for initial Ca2+ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA2 by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са2+ stores engaged by CMZ were entirely dependent on thapsigargin. The oscillations existed in the presence of inhibitors of ryanodine or inositol 1,4,5-trisphosphate receptors, or antagonists of Ca2+ transport by lysosome-like acidic stores. Carbenoxolone or octanol, blockers of hemichannels (connexons), when applied for two hours, prevented oscillations but did not affect the initial Са2+ release. Incubation with La3+ for 2 or 24 h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca2+ rise. These results suggest that Ca2+-CaM regulation suppresses La3+-sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca2+ oscillations, and connexons are intimately implicated in their generation mechanism.

Fast changes of NMDA and AMPA receptor activity under acute hyperammonemia in vitro.

It was established in experiments on cell cultures of neurons and astrocytes that ammonium ions at concentrations of 4-8 mM cause hyperexcitation of the neuronal network, as a result of which there is a disturbance of calcium homeostasis, which can lead to the death of neurons. In the present study, we investigated the effect of toxic doses of ammonium (8 mM NH4Cl) on the activity of NMDA and AMPA receptors and the role of these receptors in spontaneous synchronous activity (SSA). In a control experiment in the absence of NH4Cl, SSA is not suppressed by NMDA receptor inhibitors, but is suppressed by AMPA receptor antagonists. In the presence of toxic doses of NH4Cl, SSA is completely inhibited by NMDA receptor inhibitors in 63% of neurons and by AMPA receptor inhibitors in 33% of neurons. After short-term applications of toxic doses of ammonium, the amplitude of the Ca2+ response to 10 µM NMDA increases, and decreases in response to 500 nM FW (agonist of AMPA receptors). NMDA receptor blocker MK-801 (20 µM), competitive antagonist D-AP5 (10 µM) and competitive AMPA receptor antagonist NBQX (2 µM) abolished the activating ammonium mediated effect on the NMDA receptors while only MK-801, but not NBQX, abolished the inhibiting ammonium mediated effect on AMPA receptors. These data indicate that under acute hyperammonemia, the activity of NMDA receptors increases, while the activity of AMPA receptors decreases. This phenomenon could explain such a wide range of toxic effects of ammonium ions mediated by NMDA receptors.

Mutation in the Sip1 transcription factor leads to a disturbance of the preconditioning of AMPA receptors by episodes of hypoxia in neurons of the cerebral cortex due to changes in their activity and subunit composition. The protective effects of interleukin-10.

The Sip1 mutation plays the main role in pathogenesis of the Mowat-Wilson syndrome, which is characterized by the pronounced epileptic symptoms. Cortical neurons of homozygous mice with Sip1 mutation are resistant to AMPA receptor activators. Disturbances of the excitatory signaling components are also observed on such a phenomenon of neuroplasticity as hypoxic preconditioning. In this work, the mechanisms of loss of the AMPA receptor's ability to precondition by episodes of short-term hypoxia were investigated on cortical neurons derived from the Sip1 homozygous mice. The preconditioning effect was estimated by the level of suppression of the AMPA receptors activity with hypoxia episodes. Using fluorescence microscopy, we have shown that cortical neurons from the Sip1fl/fl mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca2+-responses to the application of the AMPA receptor agonist, 5-Fluorowillardiine, in neurons from the Sip1 mice brainstem is suppressed by brief episodes of hypoxia. The mechanism responsible for this process is hypoxia-induced desensitization of the AMPA receptors, which is absent in the cortex neurons possessing the Sip1 mutation. However, the appearance of preconditioning in these neurons can be induced by phosphoinositide-3-kinase activation with a selective activator or an anti-inflammatory cytokine interleukin-10.

Interaction of misfolded proteins and mitochondria in neurodegenerative disorders.

The number of the people affected by neurodegenerative disorders is growing dramatically due to the ageing of population. The major neurodegenerative diseases share some common pathological features including the involvement of mitochondria in the mechanism of pathology and misfolding and the accumulation of abnormally aggregated proteins. Neurotoxicity of aggregated ß-amyloid, tau, α-synuclein and huntingtin is linked to the effects of these proteins on mitochondria. All these misfolded aggregates affect mitochondrial energy metabolism by inhibiting diverse mitochondrial complexes and limit ATP availability in neurones. ß-Amyloid, tau, α-synuclein and huntingtin are shown to be involved in increased production of reactive oxygen species, which can be generated in mitochondria or can target this organelle. Most of these aggregated proteins are capable of deregulating mitochondrial calcium handling that, in combination with oxidative stress, lead to opening of the mitochondrial permeability transition pore. Despite some of the common features, aggregated ß-amyloid, tau, α-synuclein and huntingtin have diverse targets in mitochondria that can partially explain neurotoxic effect of these proteins in different brain regions.

Sip-1 mutations cause disturbances in the activity of NMDA- and AMPA-, but not kainate receptors of neurons in the cerebral cortex.

Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b), Zeb2] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome (MWS) in humans. MWS is a rare genetic autosomal dominant disease caused by a mutation in the Sip1 gene (aka Zeb2 or Zfhx1b) mapped to 2q22.3 locus. MWS affects 1 in every 50-100 newborns worldwide. It is characterized by mental retardation, small stature, typical facial abnormalities as well as disturbances in the development of the cardio-vascular and renal systems as well as some other organs. Sip1 mutations cause abnormal neurogenesis in the brain during development as well as susceptibility to epileptic seizures. In the current study we investigated the role of the Sip1 gene in the activity of NMDA-, AMPA- and KA- receptors. We showed that a particular Sip1 mutation in the mouse causes changes in the activity of both NMDA- and AMPA- receptors in the neocortical neurons in vitro. We demonstrate that neocortical neurons that have only one copy of Sip1 (heterozygous, Sip1fI/wt), are more sensitive to both NMDA- and AMPA- receptors agonists as compared to wild type neurons (Sip1wt/wt). This is reflected in higher amplitudes of agonist induced Ca2+ signals as well as a lower half maximal effective concentration (ЕC50). In contrast, neurons from homozygous Sip1 mice (Sip1fI/fI), demonstrate higher resistance to these respective receptor agonists. This is reflected in lower amplitudes of Ca2+-responses and so a higher concentration of receptor activators is required for activation.

Cytokine IL-10, activators of PI3-kinase, agonists of α-2 adrenoreceptor and antioxidants prevent ischemia-induced cell death in rat hippocampal cultures.

In the present work we compared the protective effect of anti-inflammatory cytokine IL-10 with the action of a PI3-kinase selective activator 740 Y-P, selective agonists of alpha-2 adrenoreceptor, guanfacine and UK-14,304, and compounds having antioxidant effect: recombinant human peroxiredoxin 6 and B27, in hippocampal cell culture during OGD (ischemia-like conditions). It has been shown that the response of cells to OGD in the control includes two phases. The first phase was accompanied by an increase in the frequency of spontaneous synchronous Ca2+-oscillations (SSCO) in neurons and Ca2+-pulse in astrocytes. Spontaneous Ca2+ events in astrocytes during ischemia in control experiments disappeared. The second phase started after a few minutes of OGD and looked like a sharp/avalanche, global synchronic (within 20 s) increase in [Ca2+]i in many cells. Within 1 h after OGD, a mass death of cells, primarily astrocytes, was observed. To study the protective action of the compounds, cells were incubated in the presence of the neuroprotective agents for 10-40 min or 24 h before ischemia. All the neuroprotective agents delayed a global [Ca2+]i increase during OGD or completely inhibited this process and increased cell survival.

Intracellular pH Modulates Autophagy and Mitophagy.

The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control.

Angiotensin II activates different calcium signaling pathways in adipocytes.

Angiotensin II (Ang II) is an important mammalian neurohormone involved in reninangiotensin system. Ang II is produced both constitutively and locally by RAS systems, including white fat adipocytes. The influence of Ang II on adipocytes is complex, affecting different systems of signal transduction from early Са(2+) responses to cell proliferation and differentiation, triglyceride accumulation, expression of adipokine-encoding genes and adipokine secretion. It is known that white fat adipocytes express all RAS components and Ang II receptors (АТ1 and АТ2). The current work was carried out with the primary white adipocytes culture, and Са(2+) signaling pathways activated by Ang II were investigated using fluorescent microscopy. Са(2+)-oscillations and transient responses of differentiated adipocytes to Ang II were registered in cells with both small and multiple lipid inclusions. Using inhibitory analysis and selective antagonists, we now show that Ang II initiates periodic Са(2+)-oscillations and transient responses by activating АТ1 and АТ2 receptors and involving branched signaling cascades: 1) Ang II → Gq → PLC → IP3 → IP3Rs → Ca(2+) 2) Gßγ → PI3Kγ → PKB 3) PKB → eNOS → NO → PKG 4) CD38 → cADPR → RyRs → Ca(2+) In these cascades, AT1 receptors play the leading role. The results of the present work open a perspective of using Ang II for correction of signal resistance of adipocytes often observed during obesity and type 2 diabetes.

To Break or to Brake Neuronal Network Accelerated by Ammonium Ions?

PURPOSE: The aim of present study was to investigate the effects of ammonium ions on in vitro neuronal network activity and to search alternative methods of acute ammonia neurotoxicity prevention. METHODS: Rat hippocampal neuronal and astrocytes co-cultures in vitro, fluorescent microscopy and perforated patch clamp were used to monitor the changes in intracellular Ca2+- and membrane potential produced by ammonium ions and various modulators in the cells implicated in neural networks. RESULTS: Low concentrations of NH4Cl (0.1-4 mM) produce short temporal effects on network activity. Application of 5-8 mM NH4Cl: invariably transforms diverse network firing regimen to identical burst patterns, characterized by substantial neuronal membrane depolarization at plateau phase of potential and high-amplitude Ca2+-oscillations; raises frequency and average for period of oscillations Ca2+-level in all cells implicated in network; results in the appearance of group of «run out¼ cells with high intracellular Ca2+ and steadily diminished amplitudes of oscillations; increases astrocyte Ca2+-signalling, characterized by the appearance of groups of cells with increased intracellular Ca2+-level and/or chaotic Ca2+-oscillations. Accelerated network activity may be suppressed by the blockade of NMDA or AMPA/kainate-receptors or by overactivation of AMPA/kainite-receptors. Ammonia still activate neuronal firing in the presence of GABA(A) receptors antagonist bicuculline, indicating that «disinhibition phenomenon¼ is not implicated in the mechanisms of networks acceleration. Network activity may also be slowed down by glycine, agonists of metabotropic inhibitory receptors, betaine, L-carnitine, L-arginine, etc. CONCLUSIONS: Obtained results demonstrate that ammonium ions accelerate neuronal networks firing, implicating ionotropic glutamate receptors, having preserved the activities of group of inhibitory ionotropic and metabotropic receptors. This may mean, that ammonia neurotoxicity might be prevented by the activation of various inhibitory receptors (i.e. by the reinforcement of negative feedback control), instead of application of various enzyme inhibitors and receptor antagonists (breaking of neural, metabolic and signaling systems).

The mitochondrion as a key regulator of ischaemic tolerance and injury.

Vascular pathologies pose a significant health problem because of their wide prevalence and high impact on the rate of mortality. Blockade of blood flow in major blood vessels leads to ischaemia associated with oxidative stress, where mitochondria act as a major source of reactive oxygen species (ROS). While low levels of ROS perform a necessary function in normal cellular signalling and metabolism, elevated levels under pathological conditions are detrimental both at the cell and organ level. While cellular oxygenation is necessary to maintain tissue viability, a key pathological occurrence when restoring blood flow to ischaemic tissues is the subsequent burst of ROS generation following reoxygenation, resulting in a cascade of ROS-induced ROS release. This oxygen 'paradox' is a constraint in clinical practice, that is, the need for rapid and maximal restoration of blood flow while at the same time minimising the harmful impact of reperfusion injury on damaged tissues. Mitochondria play a central role in many signalling pathways, including cardioprotection against ischaemic injury and ROS signalling, thus the main target of any anti-ischaemic protective or post-injury therapeutic strategy should include mitochondria. At present, one of the most effective strategies that provide mitochondrial tolerance to ischaemia is ischaemic preconditioning. In addition, pharmacological preconditioning which mimics intrinsic natural protective mechanisms has proven effective at priming biological mechanisms to confront ischaemic damage. This review will discuss the role of mitochondria in contributing to acute ischaemia-reperfusion (IR) injury, and mechanisms of cardioprotection in respect to mitochondrial signalling pathways.