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Gi-coupled receptors, particularly cannabinoid receptors (CBRs), are considered perspective targets for treating brain pathologies, including epilepsy. However, the precise mechanism of the anticonvulsant effect of the CBR agonists remains unknown. We have found that WIN 55,212-2 (a CBR agonist) suppresses the synchronous oscillations of the intracellular concentration of Ca 2+ ions (epileptiform activity) induced in the neurons of rat hippocampal neuron-glial cultures by bicuculline or NH 4 Cl. As we have demonstrated, the WIN 55,212-2 effect is mediated by CB 1 R receptors. The agonist suppresses Ca 2+ inflow mediated by the voltage-gated calcium channels but does not alter the inflow mediated by NMDA, AMPA, and kainate receptors. We have also found that phospholipase C (PLC), protein kinase C (PKC), and G-protein-coupled inwardly rectifying K + channels (GIRK channels) are involved in the molecular mechanism underlying the inhibitory action of CB 1 R activation against epileptiform activity. Thus, our results demonstrate that the antiepileptic action of CB 1 R agonists is mediated by different intracellular signaling cascades, including non-canonical PLC/PKC-associated pathways.

This Commentary presents a brief discussion of the action of glutamate calcium permeable receptors present with neurons on the release of the neurotransmitter gamma-aminobutyric acid (GABA). In particular, Glutamate sensitive Kainic Acid Receptors (KARs) and α-Amino-3-hydroxy-5-Methyl-4-isoxazole Propionic Acid Receptor (AMPARs) are Na + channels that typically cause neuronal cells to depolarize and release GABA. Some of these receptors are also permeable to Ca 2+ and are hence involved in the calcium-dependent release of GABA neurotransmitters. Calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs) are predominantly located in GABAergic neurons in the mature brain and their primary role is to regulate GABA release. AMPARs which do not contain the GluA2 subunit are mainly localized in the postsynaptic membrane. CP-KAR receptors are located mainly in the presynapse. GABAergic neurons expressing CP-KARs and CP-AMPARs respond to excitation earlier and faster, suppressing hyperexcitation of other neurons by the advanced GABA release due to an early rapid [Ca 2+ ] i increase. CP-AMPARs have demonstrated a more pronounced impact on plasticity compared to NMDARs because of their capacity to elevate intracellular Ca 2+ levels independently of voltage. GABAergic neurons that express CP-AMPARs contribute to the disinhibition of glutamatergic neurons by suppressing GABAergic neurons that express CP-KARs. Hence, the presence of glutamate CP-KARs and CP-AMPARs is crucial in governing hyperexcitation and synaptic plasticity in GABAergic neurons., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

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 Ca 2+ influx. Here, we demonstrate that neurons containing CP-AMPARs are characterized by the higher amplitude of the glutamate-induced elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) and slower restoration of [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] 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 Ca 2+ influx and slow removal of Ca 2+ 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate in vitro called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca 2+ concentration ([Ca 2+ ] i ). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture., Methods: A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca 2+ ] i level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca 2+ ] i imaging., Results and Discussion: It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca 2+ ] i pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca 2+ pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zinchenko, Teplov, Kosenkov, Gaidin, Kairat and Tuleukhanov.)

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 Ca 2+ 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 G i -coupled receptors, such as A 1 adenosine (A 1 Rs) or cannabinoid receptors (CBRs) (N 6 -cyclohexyladenosine and WIN 55,212-2, respectively), completely suppressed PDS generation, while the A 1 R agonist even prevented it. We hypothesized that the dynamics of extracellular glutamate concentration govern paroxysmal activity. Fine-tuning of neuronal activity via action on G i -coupled receptors or iGluRs paves the way for the development of new approaches for epilepsy pharmacotherapy.

The effect of the compound N 1 -(2,3,4-trimethoxy)-N 2 -{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca 2+ response in the cell was studied in in vitro experiments. The concentration of intracellular calcium was assessed using a Fura-2 two-wave probe. The experiments were performed on a culture of isolated rat hippocampal neurons. The effect of compound ALM-802 on the activity of ryanodine receptors (RyR2) was studied on an isolated strip of rat myocardium. The compound ALM-802 (69.8 μM) in hippocampal neurons causes a significant decrease in the amplitude of the Ca 2+ response induced by addition of KCl to the medium. Experiments performed on an isolated myocardial strip showed that compound ALM-802 (10 -5 M) almost completely blocked the positive inotropic reaction of the strip to the RyR2 agonist caffeine (5×10 -5 M). The data obtained indicate that the decrease in the concentration of Ca 2+ ions in the cell caused by ALM-802 is due to its ability to block RyR2 located on the membrane of the sarcoplasmic reticulum, which can be associated with the antiarrhythmic activity of the compound., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

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 Ca 2+ influx in neurons during activation of AMPARs in the presence of NMDAR and KAR antagonists, and blockers of voltage-gated Ca 2+ 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 Ca 2+ 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., (© 2022 International Society for Neurochemistry.)

The mechanisms underlying the antiarrhythmic action of compound trihydrochloride N 1 -(2,3,4-trimethoxy)-N 2 -{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) were studied in vitro. The experiments were performed on a culture of rat hippocampal neurons. The electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It is shown that the compound ALM-802 effectively blocks potential-dependent Na + and K + channels and does not affect the activity of potential-dependent Ca 2+ channels. The inhibition of currents through these channels is dose-dependent; the IC 50 of Na + and K + channels were 94±4 and 67±3 μM, respectively. These findings indicate that compound ALM-802 combines the properties of class I and class III antiarrhythmic agents according to the Vaughan-Williams classification., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

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 Ca 2+ 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.

In in vitro experiments on isolated rat hippocampal neurons, we studied the electrophysiological mechanisms of the antiarrhythmic effects of N-deacetyllappaconitine monochlorhydrate (DALCh), active metabolite of lappaconitine hydrobromide (allapinin). Electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It was shown that DALCh increased the duration of both slow and fast depolarization phases and decreased the amplitude of the action potential. DALCh effectively inhibited transmembrane currents of Na + ions and partially K + ions through the corresponding transmembrane voltage-gated ion channels. Thus, DALCh, in contrast to lappaconitine hydrobromide, belongs not to 1C, but to the 1A class of antiarrhythmics according to the Vaughan-Williams classification., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs), as well as NMDARs, play a pivotal role in plasticity and in regulating neurotransmitter release. Here we visualized in the mature hippocampal neuroglial cultures the neurons expressing CP-AMPARs and CP-KARs. These neurons were visualized by a characteristic fast sustained [Ca 2+ ] i increase in response to the agonist of these receptors, domoic acid (DoA), and a selective agonist of GluK1-containing KARs, ATPA. Neurons from both subpopulations are GABAergic. The subpopulation of neurons expressing CP-AMPARs includes a larger percentage of calbindin-positive neurons (39.4 ± 6.0%) than the subpopulation of neurons expressing CP-KARs (14.2 ± 7.5% of CB + neurons). In addition, we have shown for the first time that NH 4 Cl-induced depolarization faster induces an [Ca 2+ ] i elevation in GABAergic neurons expressing CP-KARs and CP-AMPARs than in most glutamatergic neurons. CP-AMPARs antagonist, NASPM, increased the amplitude of the DoA-induced Ca 2+ response in GABAergic neurons expressing CP-KARs, indicating that neurons expressing CP-AMPARs innervate GABAergic neurons expressing CP-KARs. We assume that CP-KARs in inhibitory neurons are involved in the mechanism of outstripping GABA release upon hyperexcitation.

Hippocampal GABAergic neurons are subdivided into more than 20 subtypes that are distinguished by features and functions. We have previously described the subpopulation of GABAergic neurons, which can be identified in hippocampal cell culture by the calcium response to the application of domoic acid (DoA), an agonist of kainate receptors (KARs). Here, we investigate the features of DoA-sensitive neurons and their GABA release mechanism in response to KARs activation. We demonstrate that DoA-sensitive GABAergic neurons express GluK1-containing KARs because ATPA, a selective agonist of GluK1-containing receptors, induces the calcium response exclusively in these GABAergic neurons. Our experiments also show that NASPM, previously considered a selective antagonist of calcium-permeable AMPARs, blocks calcium-permeable KARs. We established using NASPM that GluK1-containing receptors of the studied population of GABAergic neurons are calcium-permeable, and their activation is required for GABA release, at least in particular synapses. Notably, GABA release occurs even in the presence of tetrodotoxin, indicating that propagation of the depolarizing stimulus is not required for GABA release in this case. Thus, our data demonstrate that the activation of GluK1-containing calcium-permeable KARs mediates the GABA release by the studied subpopulation of GABAergic neurons., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

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 Ca 2+ concentration ([Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] i level during oscillations, which appears to determine the number of PDSs in the cluster.

Spontaneous burst firing is a hallmark attributed to the neuronal network activity. It is known to be accompanied by intracellular calcium [Са 2+ ] i oscillations within the bursting neurons. Studying mechanisms underlying regulation of burst firing is highly relevant, since impairment in neuronal bursting accompanies different neurological disorders. In the present study, the contribution of NMDA and GABA(A) receptors to the shape formation of spontaneous burst -was studied in cultured hippocampal neurons. A combination of inhibitory analysis with simultaneous registration of neuronal bursting by whole-cell patch clamp and calcium imaging was used to assess spontaneous burst firing and [Са 2+ ] i level. Using bicuculline and D-AP5 we showed that GABA(A) and NMDA receptors effectively modulate burst plateau phase and [Са 2+ ] i transient spike which can further affect action potential (AP) amplitudes and firing frequency within a burst. Bicuculline significantly elevated the amplitude and reduced the duration of both burst plateau phase and [Са 2+ ] i spike resulting in an increase of AP firing frequency and shortening of AP amplitudes within a burst. D-AP5 significantly decreases the amplitude of both plateau phase and [Са 2+ ] i spike along with a burst duration that correlated with an increase in AP amplitudes and reduced firing frequency within a burst. The effect of bicuculline was occluded by co-addition of D-AP5 revealing modulatory role of GABA(A) receptors to the NMDA receptor-mediated formation of the burst. Our results provide new evidence on importance of NMDA and GABA(A) receptors in shaping burst firing and Ca 2+ transient spikes in cultured hippocampal neurons., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 (pH i ) 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, pH i , the intensity of superoxide production in hippocampal cells under acute hyperammonemia., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 NH 4 Cl-induced oscillations of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca 2+ ] 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 [Ca 2+ ] i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca 2+ ] i rise, mediated by G iβγ subunit and subsequent PLC-induced mobilization of Ca 2+ from internal stores, and a plateau phase, mediated by a Ca 2+ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca 2+ response in astrocytes and suppression of [Ca 2+ ] i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of G βγ -signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca 2+ ] 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 G iβγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity., (© 2019 Wiley Periodicals, Inc.)

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 Ca 2+ conductivity of ionotropic glutamate receptors and voltage-gated Ca 2+ channels changes in an in vitro model of epilepsy. For induction of epileptiform activity, hippocampal neurons were exposed to Mg 2+ -free medium. It has been shown that removal of Mg 2+ 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 Mg 2+ -free medium-induced Ca 2+ oscillations decrease after the 60-min exposure. Besides, the Ca 2+ conductivity of ionotropic glutamate receptors and voltage-gated calcium channels significantly reduces. Thus, the decrease of Ca 2+ conductivity can be considered as one of the mechanisms of adaptation during epilepsy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Intracellular Са 2+ controls its own level by regulation of Ca 2+ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca 2+ concentration in different cells. We have found persistent Са 2+ 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 Ca 2+ and were supported by Са 2+ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A 2 inhibited by AACOCF 3 was responsible for initial Ca 2+ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA 2 by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са 2+ 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 Ca 2+ 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 Са 2+ release. Incubation with La 3+ for 2 or 24 h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca 2+ rise. These results suggest that Ca 2+ -CaM regulation suppresses La 3+ -sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca 2+ oscillations, and connexons are intimately implicated in their generation mechanism.

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) IP 3 R, 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.

Cerebral blood flow disturbances lead to the massive death of brain cells. The death of >80% of cells is observed in hippocampal cell cultures after 40 min of oxygen and glucose deprivation (ischemia-like conditions, OGD). However, there are some populations of GABAergic neurons which are characterized by increased vulnerability to oxygen-glucose deprivation conditions. Using fluorescent microscopy, immunocytochemical assay, vitality tests and PCR-analysis, we have shown that population of GABAergic neurons are characterized by a different (faster) Ca 2+ dynamics in response to OGD and increased basal ROS production under OGD conditions. A plant flavonoid taxifolin inhibited an excessive ROS production and an irreversible cytosolic Ca 2+ concentration increase in GABAergic neurons, preventing the death of these neurons and further excitation of a neuronal network; neuroprotective effect of taxifolin increased after incubation of 24 h and correlated with increased expression of antiapoptocic and antioxidant genes Stat3 Nrf-2 Bcl-2, Bcl-xL, Ikk2, and genes coding for AMPA and kainate receptor subunits; in addition, taxifolin decreased expression of prooxidant enzyme NOS and proinflammatory cytokine IL-1β., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effect of a cerebroprotective agent magnesium bis-aminoethanesulfonate (laboratory code FS-LKhT-317) on intracellular calcium concentration was studied by the fluorescent imaging technique on neuroglial cell culture from Spraque-Dawley rat hippocampus. The substance produced a pronounced inhibitory effect and suppressed NMDA receptor activity in concentrations of ≥50 μM. The observed effects were reversible or partially reversible and were detected by a decrease in Ca 2+ signal amplitude in neurons in response to NMDA applications in a Mg 2+ -free medium and by inhibition of Ca 2+ pulses in magnesium-free medium (elimination of magnesium block).

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 NH 4 Cl) 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 NH 4 Cl, SSA is not suppressed by NMDA receptor inhibitors, but is suppressed by AMPA receptor antagonists. In the presence of toxic doses of NH 4 Cl, 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 Ca 2+ 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 Sip1 fl/fl mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca 2+ -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., (Copyright © 2018. Published by Elsevier Inc.)

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 β-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. β-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 β-amyloid, tau, α-synuclein and huntingtin have diverse targets in mitochondria that can partially explain neurotoxic effect of these proteins in different brain regions., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

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, Sip1 fI/wt ), are more sensitive to both NMDA- and AMPA- receptors agonists as compared to wild type neurons (Sip1 wt/wt ). This is reflected in higher amplitudes of agonist induced Ca 2+ signals as well as a lower half maximal effective concentration (ЕC50). In contrast, neurons from homozygous Sip1 mice (Sip1 fI/fI ), demonstrate higher resistance to these respective receptor agonists. This is reflected in lower amplitudes of Ca 2+ -responses and so a higher concentration of receptor activators is required for activation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 Ca 2+ -oscillations (SSCO) in neurons and Ca 2+ -pulse in astrocytes. Spontaneous Ca 2+ 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 [Ca 2+ ] 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 [Ca 2+ ] i increase during OGD or completely inhibited this process and increased cell survival., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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) Gβγ → 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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 out» 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 phenomenon» 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 ratio of early apoptosis and late apoptosis (necrosis) in the cultured human umbilical vein endothelial cells was estimated after exposure to hydrogen peroxide (H2O2) in vitro trying to keep them close to the physiological conditions (high cell density, high serum content, H2O2 concentration not over 500 μM). Cell viability was assessed using flow cytometry and simultaneous staining with fluorescent dyes PO-PRO-1 to detect early apoptotic cells, and DRAQ7 to detect late apoptotic and necrotic cells. The data obtained suggest that the primary mechanism of cytotoxic response is apoptosis. The critical concentration of H2O2 causing the death of the cell population in a dense monolayer is 250 μM. Lower concentrations of H2O2 (up to 200 μM) cause death of individual cells; however, viability of endothelial cell population is retained, and response to calcium activating agonists does not change compared with control cells.

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., (Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)

It is suggested that anti-inflammatory cytokine interleukin-10 (IL-10) mediates the delayed protective effects through activation of Jak-Stat3, PI3K-Akt and NF-κB signaling pathways. However, our previous experiments have demonstrated that IL-10 is capable to exert the rapid neuroprotective action through modulation of hypoxia-induced intracellular Ca(2+) ([Ca(2+)]i) response. The first purpose of the present study was to evaluate the neuroprotective effects of IL-10 using three models of the ischemic insults in rats: permanent middle cerebral artery occlusion, ischemia in acute hippocampal slices in vitro and ischemia in cultured hippocampal cells in vitro. The second purpose of the study was to elucidate a role of [Ca(2+)]i changes in the mechanisms underlying IL-10 elicited protection of neurons and astrocytes from ischemia-induced death in cultures of primary hippocampal cells. The data presented here shown that anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats. This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We have previously developed a rat hippocampal neuronal cell model for the registration of the preconditioning effect and posthypoxic hyperexcitability (Turovskaya et al., 2011). Repeated episodes of short-term hypoxia are reported to suppress the amplitude of Ca(2+) response to NMDA in majority of neurons, reflecting the effect of preconditioning in the culture. In addition, exposure to hypoxia causes posthypoxic hyperexcitability: this is characterized by the onset of spontaneous synchronous Ca(2+) transients in a population of neurons in a neural network during the period of reoxygenation after each hypoxic episode. The nature of this phenomenon is unknown, although it has been observed that there always exists a minority of neurons in which there is no effect of hypoxic preconditioning. In this small population of neurons, the amplitude of Ca(2+) response to NMDA is not suppressed, but rather increases after each episode of hypoxia. Here we report the type of these neurons and their role in the generation of posthypoxic hyperexcitability. We compared the effect of short-term hypoxia on the amplitude of the Ca(2+) response to NMDA and the Ca(2+) transient generation in two populations of neurons - inhibitory GABAergic and excitatory glutamatergic. We have demonstrated that the neurons in which the preconditioning effect was not observed are GABAergic. Moreover at the instant moment of the posthypoxic synchronous Ca(2+)-transient generation (during reoxygenation) there is a global increase of [Ca(2+)]i and subsequent apoptosis in some GABAergic neurons. Anti-inflammatory cytokine interleukin-10 prevents the development of posthypoxic hyperexcitability, inhibiting the spontaneous synchronous Ca(2+) transients. At the same time, interleukin-10 protects GABAergic neurons from death, by restoring the effect of hypoxic preconditioning in them. Activation of one of the signaling pathways initiated by interleukin-10 appears to be necessary for the development of hypoxic preconditioning in GABAergic neurons. Overall our results indicate that short-term episodes of hypoxia can damage GABAergic neurons and weaken the inhibitory action of GABAergic neurons in a neural network. Activation of PI3K-dependent survival signaling pathways in neurons of this type is a possible strategy to protect these cells against hypoxia., (© 2013.)

Purpose: This study investigated possible mechanisms of autoregulation of Ca(2+) signalling pathways in adipocytes responsible for Ca(2+) and NO oscillations and switching phenomena promoted by acetylcholine (ACh), norepinephrine (NE) and atrial natriuretic peptide (ANP)., Methods: Fluorescent microscopy was used to detect changes in Ca(2+) and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca(2+)-channels in Ca(2+) signalling pathways., Results: ACh activating M3-muscarinic receptors and Gβγ protein dependent phosphatidylinositol 3 kinase induces Ca(2+) and NO oscillations in adipocytes. At low concentrations of ACh which are insufficient to induce oscillations, NE or α1, α2-adrenergic agonists act by amplifying the effect of ACh to promote Ca(2+) oscillations or switching phenomena. SNAP, 8-Br-cAMP, NAD and ANP may also produce similar set of dynamic regimes. These regimes arise from activation of the ryanodine receptor (RyR) with the implication of a long positive feedback loop (PFL): Ca(2+)→NO→cGMP→cADPR→Ca(2+), which determines periodic or steady operation of a short PFL based on Ca(2+)-induced Ca(2+) release via RyR by generating cADPR, a coagonist of Ca(2+) at the RyR. Interplay between these two loops may be responsible for the observed effects. Several other PFLs, based on activation of endothelial nitric oxide synthase or of protein kinase B by Ca(2+)-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca(2+)-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G., Conclusions: This study presents a kinetic model of Ca(2+)-signalling system operating in adipocytes and integrating signals from various agonists, which describes it as multivariable multi feedback network with a family of nested positive feedback.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Metabotropic serotonin receptors of 5HT1-type in brain neurons participate in regulation of such human emotional states as aggression, fear and dependence on alcohol. Activated presynaptic 5-HT1B receptors suppress the Ca2+ influx through the potential-dependent calcium channels in certain neurons. The Ca2+ influx into the cells has been measured by increase of calcium ions concentration in cytoplasm in reply to the depolarization caused by 35mM KC1. Using system of image analysis in hippocampal cells culture we found out that Ca2+-signals to depolarization oin various populations of neurons differed in form, speed and amplitude. 5HT1B receptor agonists in 86 +/- 3 % of neurons slightly suppressed the activity of potential-dependent calcium channels. Two minor cell populations (5-8 % of cells each) were found out, that strongly differed in Ca2+ signal desensitization. Calcium signal caused by depolarization in one cells population differed in characteristic delay and high rate of decay. 5HT1B receptor agonists strongly inhibited the amplitude of the Ca2+ response on KCl only in this population of neurons. The calcium signal in second cell population differed by absence desensitization and smaller amplitude which constantly increased during depolarization. 5HT 1 B receptor agonists increased the calcium response amplitude to depolarization in this population of neurons. Thus we show various sensitivity of potential-dependent calcium channels of separate neurons to 5HTB1 receptor agonist.

Using selective receptor's agonist and antagonists we show that mouse white fat cells express alpha1A-, alpha2-adrenergic receptors, which activation with noradrenaline is capable of causing calcium responses different by formation mechanism. Adipocyte's calcium responses to alpha1-adrenoreceptor agonists are caused by alpha1A-type adrenoreceptor and suppressed by inhibitors of PLC-dependent pathway. Calcium responses to alpha2-adrenoreceptors agonists are realized only in the presence of more than 200 microM of L-arginine and suppressed by inhibitors of NOS-PKG-RyR pathway. The incubation of cells with L-arginine creates conditions for switching on the signal pathway with participation of eNOS --> NO --> sGC --> cGMP --> PKG --> CD38 --> RyR --> Ca2+ and for switching of the PLC - IP3R-dependent pathway. Adipocyte's calcium response to L-arginine represents a sharp impulse of the big amplitude and is mediated by alpha2-adrenoreceptors. L-arginine activating alpha2-adrenoreceptors and being the substrate of eNOS, realizes two functions in this pathway.

The goal of this study was to evaluate an effect of interleukin-10 (IL-10) on the Ca(2+) response induced by repeated NMDA receptor activation with brief hypoxia in cultured hippocampal neurons. We focused on the importance of internal Ca(2+) stores in the modulation of this Ca(2+) response by IL-10. To test this, we compared roles of InsP(3)- and ryanodine-sensitive internal stores in the effects of IL-10. Measurements of intracellular cytosolic calcium concentration ([Ca(2+)](i)) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. Repeated episodes of NMDA receptor activation with brief hypoxia induced the spontaneous (s) [Ca(2+)](i) increases about 3 min after each hypoxic episode. The amplitude of the s[Ca(2+)](i) increases was progressively enhanced from the first hypoxic episode to the third one. IL-10 (1 ng/ml) abolished these s[Ca(2+)](i) increases. Exposure of cultured hippocampal neurons with thapsigargin (1 μM) or an inhibitor of phospholipase C (U73122, 1 μM) for 10 min also abolished the s[Ca(2+)](i) increases. On the other hand, antagonist of ryanodine receptors (ryanodine, 1 μM) did not affect this Ca(2+) response. These studies appear to provide the first evidence that Ca(2+) release from internal stores is affected by anti-inflammatory cytokine IL-10 in brain neurons. It is suggested that these data increase our understanding of the neuroprotective mechanisms of IL-10 in the early phase of hypoxia., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

A method for the detection and characterization of GABA(A) receptors of neurons has been developed, which is based on the measurement of the activity of potential-dependent calcium channels using the fluorescence of the two-wavelength calcium-sensitive probe Fura-2. The method makes it possible to detect the ligands of GABA(A) receptors and determine the constants of activation and inhibition as well as the type of inhibition. The object of investigation was a young (two- to four-day-old) rat hippocampal cell culture in which GABA induces the depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to the activation of potential-dependent calcium channels. It was shown that a short-time application of GABA induces a decrease in the amplitude of calcium responses to subsequent addition of the depolarizing agents GABA or KCl. However, at low amplitudes of calcium responses to the addition of GABA, this reducing effect on the subsequent addition of KCl was insignificant. It was found that the amplitudes of calcium responses to KCl and GABA are linearly dependent on the angular coefficient b = 3.41. This enabled one to develop a method of normalizing calcium signals, which makes it possible to compare experiments performed on different days and different cultures. By using this normalization technique, the values of EC50 = 2.21 +/- 0.14 ?M and the Hill coefficient = 1.9 +/- 0.2 were estimated. The blocker of potential-dependent calcium channels nifedipine suppressed simultaneously the amplitudes of calcium responses to the addition of KCl and GABA. In this case, the linear relationship between the amplitudes of calcium responses to the addition of KCl and GABA was retained. To verify the validity of the method, the constant of inhibition of a calcium signal and the type of inhibition for known noncompetitive and competitive antagonists of GABA(A) receptors were determined.

The aim of this study was to evaluate the intracellular cytosolic calcium concentration ([Ca(2+)](i)) changes induced by activation of ionotropic glutamate receptors in cultured hippocampal neurons after repeated brief episodes of hypoxia. To investigate what kinds of ionotropic glutamate receptors are involved we used specific agonists for AMPA- and NMDA-type glutamate receptors. Measurements of [Ca(2+)](i) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. In the rat hippocampal slice method, field potential measurements in CA1 pyramidal neurons were used. The main result of our study is that brief hypoxic episodes progressively depress the [Ca(2+)](i) increases induced by agonists of AMPA and NMDA glutamate receptors in cultured hippocampal neurons. An effectiveness of this depression is increased from the first hypoxic episode to the third one. Hypoxic preconditioning effect is observed during 10-20 min after termination of hypoxic episode and depends on [Ca(2+)](i) response amplitudes to agonists before hypoxia. In contrast to AMPA receptor activation, NMDA receptor activation before hypoxia induce the spontaneous [Ca(2+)](i) increase about 3 min after each hypoxic episode. These spontaneous [Ca(2+)](i) increases may be an indicator of the development of posthypoxic hyperexcitability in hippocampal neurons. Our results suggest that brief hypoxia-induced depression of the glutamate receptor-mediated [Ca(2+)](i) responses contributes to the development of rapid hypoxic preconditioning in hippocampal CA1 neurons., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The biological properties of dihydroquercetin (DHO) modified by including it into the ring of beta-cyclodextrin (beta-CD) to give it more water-soluble properties have been investigated. It was shown that the peroral administration of the DHQ/beta-CD complex provides a long increase of DHQ concentration in rat blood (up to 7.5 h), and, unlike pure DHQ, the complex does not accumulate in the liver. As DHQ is released from the complex, it penetrates into liposome membranes, changing their thermodynamic characteristics. DHQ decreases the specific heat absorption, enthalpies, and temperature maximum of lipid melting and increases the transition half-width. This property is used to estimate the stability of the DHQ/beta-CD complex. It was shown that complex DHQ/beta-CD is not stable, and DHQ molecules slowly leave the complex in water environment. Seven and a half hours after the peroral injection of drugs, DHQ was found in the blood plasma of rats to which water-soluble complex DHQ/betaCD was injected and in the liver of rats to which free DHQ was injected. Thus, DHQ/betaCD not only is a more water-soluble complex but also it slowly releases DHQ, supporting long a low concentration of the free form of DHQ and providing the penetration of DHQ into the blood stream. After several weeks of feeding old mice with antioxidants, the activity of mitochondrial enzymes was restored to the level observed in young animals.

Thermogenic capability of brown adipose tissue is controlled by norepinephrine. Interaction of norepinephrine with adipocyte at- and P3-adrenergic receptors results in the increase of Ca2+ and cAMP concentrations. The [Ca2+]i changes initiated by norepinephrine and selective agonists of alpha1- and beta-adrenergic receptors, cirazolin and isoproterenol, were recorded in single cells of primary culture on the 1st, 3rd and 6th days in vitro. On the first day, isoproterenol-induced [Ca2+]i changes as compared to cirazolin-induced ones were characterized by greater amplitude and lesser impulse duration over the entire range of physiological concentrations used. These differences were negligible after 3 days and kinetic differences were practically absent after 6 days of cultivation. The agonist-induced [Ca2+]i changes in proliferating and differentiated cells differed significantly: in the process of cell growth in culture, the amplitude of calcium response increased, the duration of impulse signal decreased and the sensitivity to adrenergic agonists increased. The Ca2+ store in endoplasmic reticulum increased during the cell growth and development in culture, according to thapsigargin-induced Ca2+ response amplitude increase in Ca2+ free medium. The rate of Ca2+ pumping out of cell characterizing PMCA-activity also increased.

It has been shown using the fluorescent microscopy technique that long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, exert the most toxic effect on rat ventricular cardiomyoctes. The addition of 20-50 microM acylcarnitines increases calcium concentration in cytoplasm ([Ca2+]i) and causes cell death after the 4-8 min lag-period. This effect is independent on extracellular calcium and L-type calcium channel inhibitors. Free acids (myristic and palmitic acids) at a concentration of 300-500 microM have a little effect on [Ca2+]i within 30 min. We suggest that the toxic effect is due to the activation of sarcoplasmic reticulum calcium channels by acylcarnitines and resulting acyl-CoA. Mitochondria play a role of calcium-buffer system in these conditions. The calcium capacity of this buffer determines the lag-period. Phosphate increases the calcium capacity of mitochondrial and the lag-period. In the presence of rotenone and oligomycin the elevation of [Ca2+]i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondrial leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.

Analysis of the slow Ca(2+)-responses of brown preadipocytes of ground squirrel Spermophillus undulatus and mouse was carried out. The mouse brown preadipocytes demonstrated low but prominent responses to noradrenalin with the maximum at 3 and 10 microM being the less effective. The ground squirrel brown preadipocytes practically did not practically respond to 10 nM-10 microM, whereas 30-600 microM noradrenalin was able to raise intracellular [Ca2+]i up to 600 nM with 300 microM agonist being the most effective. Stimulation of the plasma membrane Ca(2+)-channels with thimerosal showed considerable reduction of the calcium entry system in the cell precursors of both species comparing with their mature adipocytes. Intracellular calcium stores liberated in preadipocytes of both species by tapsigargin and ionomycin in Ca(2+)-free medium were insignificant, and capacitative Ca(2+)-entry in response to the cellular Ca(2+)-stores depletion was completely absent in Ca(2+)-containing medium. The Ca(2+)-responses of the ground squirrel brown preadipocytes were independent on physiological state of the animals and annual seasons. Preadipocytes of both species showed the same dose-response curves for the Ca(2+)-raise under thimerosal, and the mouse had two-fold higher kinetic constants for the Ca2+ ions entry. The ground squirrel brown adipocytes responded to ionomycin with approximately 25% higher increase in [Ca2+]i and the entry of the ions had 7-10-fold higher kinetic constants for this process. Kinetic constants for the [Ca2+]i raise in mouse preadipocytes were independent of ionomycin concentration, whereas in the ground squirrel brown preadipocytes the constant linearly increased with the ionophore concentration. It is suggested that the found difference in the function of Ca(2+)-signalling in preadipocytes of two species, which becomes apparent in the presence of ionomycin, might be responsible for the observed difference in the noradrenalin induced cellular Ca(2+)-responses as well.

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.

The properties of the Ca2+ channel induced by a calmodulin inhibitor in Ehrlich ascites tumor cells were investigated using fluorescent indicators Indo-1 and chlortetracycline. The inhibitor of calmodulin calmidazolium (R24571) in concentrations of 1-2 microM induces a short-term Ca2+ entry and a pulse-like ATP secretion. Repeated addition of R24571 also causes a transient Ca2+ signal. Ca2+ channels induced by R24571 are permeable for Mn2+. Ca2+ entry does not depend on endoplasmic reticulum depletion by thapsigargin, ATP, or ionomycin and is suppressed by nordihydroguaretic acid (EC50 = 6.7 microM), quercetin (EC50 = 1.5 microM), dihydroquercetin (EC50 = 17 microM), arachidonic acid (AA) (EC50 = 8.6 microM), and suramin (EC50 = 0.25 +/- 0.05 MM), and weakly depends on temperature in the range of 18 - 37 degrees C. The apparent activation constant for R24571 and the Hill coefficient are 2.5 +/- 0.2 and 4 +/- 0.3 microM, respectively. The products of arachidonic acid oxidation are neither activators nor inhibitors of these channels. The inhibitory effect of nordihydroguaretic acid is indirect and is conceivably caused by the accumulation of arachidonic acid due to suppression of its lipoxygenase-catalyzed oxidation at phospholipase A2 activation. The maximal level of about 1.3 microM in the dependence of Ca2+ signal amplitude on R24571 concentration points to possible inhibition of the channel by increased Ca2+ concentration in the cytosol. The weak dependence on temperature implies that the channel is highly permeable, the chain of enzymic processes is not involved in Ca2+ entry activation, and the mutual compensation of processes with opposite contributions is possible. Using chlortetracycline fluorescence, we have shown in model experiments on calmodulin solution that Ca2+ induces cooperatively a conformational transition of calmodulin with the exposure of a hydrophobic chlortetracycline-Ca(2+)-binding site. The interaction of R24571 with the CaM-Ca2+ complex results in quenching of fluorescence to its level in water, which is interpreted as the elimination of the availability of calmodulin hydrophobic site for chlortetracycline-Ca+. Nordihydroguaretic acid, quercetin, and dihydroquercetin, but not suramin, also interact with calmodulin, but this does not result in the complete closing of its hydrophobic site. It is supposed that the activation of the Ca2+ channel occurs owing to the activation of calmodulin-dependent phospholipase A2 by R24571, which leads to the formation of a low-molecular short-lived secondary messenger, or because of the interaction of R24571 with calmodulin, which directly inhibits the channel. The termination of Ca2+ entry is probably due to the inhibition of phospholipase A2 and/or of the channel at increased concentrations of arachidonic acid and Ca2+.