Egor A. Turovsky

Interleukin-10 modulates [Ca2+]i response induced by repeated NMDA receptor activation with brief hypoxia through inhibition of InsP3-sensitive internal stores in hippocampal neurons

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.

Repeated brief episodes of hypoxia modulate the calcium responses of ionotropic glutamate receptors in hippocampal neurons

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.

Anti-inflammatory cytokine interleukin-10 increases resistance to brain ischemia through modulation of ischemia-induced intracellular Ca2+ response

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.

Acetylcholine promotes Ca2+ and NO-oscillations in adipocytes implicating Ca2+→NO→cGMP→cADP-ribose→Ca2+ positive feedback loop--modulatory effects of norepinephrine and atrial natriuretic peptide.

Purpose This study investigated possible mechanisms of autoregulation of Ca2+ signalling pathways in adipocytes responsible for Ca2+ 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 Ca2+ and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca2+-channels in Ca2+ signalling pathways. Results ACh activating M3-muscarinic receptors and Gβγ protein dependent phosphatidylinositol 3 kinase induces Ca2+ 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 Ca2+ 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): Ca2+→ NO→cGMP→cADPR→Ca2+, which determines periodic or steady operation of a short PFL based on Ca2+-induced Ca2+ release via RyR by generating cADPR, a coagonist of Ca2+ 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 Ca2+-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca2+-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 Ca2+-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.

Short-term episodes of hypoxia induce posthypoxic hyperexcitability and selective death of GABAergic hippocampal neurons

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.

Convergence of Ca2+ signaling pathways in adipocytes. The role of L-arginine and protein kinase G in generation of transient and periodic Ca2+ signals

Experiments on cultured mouse adipocytes (9 days in vitro) using fluorescent microscopy have shown that activation of α1- and α2-adrenoceptors by norepinephrine (NE) or α2-adrenoreceptors by L-arginine evokes transient Ca2+ signals, while activation of m3-cholinoreceptors by acetylcholine (ACh) or betaine causes sustained or damped Ca2+ oscillations. The presence in the incubation medium of L-arginine at a low concentration (100–200 μM) is necessary for a vigorous manifestation of these effects, apparently due to transition of protein kinase G (PKG) and phosphodiesterase V into an active state. In the presence of 1–10 mM L-arginine, the amplitude of the Ca2+ transient response to NE increases and signal duration decreases. ACh and NE upon a sequential addition mutually potentiate their effects. Using an inhibitory analysis we show that the observed modes are related to the operation of a signaling pathway with the participation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), endothelial NO synthase (eNOS), cytoplasmic guanylate cyclase (sGC), protein kinase G (PKG), ADP-ribosyl cyclase (CD38), and the ryanodine receptor (RyR). The formation of several loops of positive feedbacks (PF) and negative feedbacks (NF) in the signaling system is possible: (i) short PF loops due to Ca2+-induced Ca2+ release (CICR) from internal stores through the inositol trisphosphate receptor (IP3R) and RyR participating in the transient signal formation; (ii) long PF loop Ca2+ → eNOS → sGC → PKG → CD38 → RyR → Ca2+, which can provide necessary conditions for calcium oscillations arising from short PF loops (CICR); (iii) several NF loops based on PKG-mediated inhibition of IP3R and activation of Ca2+-ATPases of sarco(endo)plasmic reticulum and of the plasma membrane providing a shutdown of signaling by the pathway phospholipase C → IP3R → Ca2+ and limiting Ca2+ rise caused by the pathway PI3K → PKB → eNOS → sGC → PKG → CD38 → RyR → Ca2+. Convergence of signaling pathways that involve α1-, α2-, and m3-receptors and then Gβγ-subunits of Gq and Gq proteins acting on PI3Kγ can provide activation of cytoplasmic PKG, which plays a key role in producing transient responses, in activation of Ca2+ removal and generation of [Ca2+]i oscillations. PKG inhibition (implemented here by KT5823 application) in the presence of any agonist results in rupture of NF loops controlling Ca2+ transporting systems activity that leads to uncontrolled [Ca2+]i rise and cell death.

Brain metabolic sensing and metabolic signaling at the level of an astrocyte

Astrocytes support neuronal function by providing essential structural and nutritional support, neurotransmitter trafficking and recycling and may also contribute to brain information processing. In this article we review published results and report new data suggesting that astrocytes function as versatile metabolic sensors of central nervous system (CNS) milieu and play an important role in the maintenance of brain metabolic homeostasis. We discuss anatomical and functional features of astrocytes that allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (oxygen and glucose), and metabolic waste products (carbon dioxide). We report data suggesting that astrocytes are also sensitive to circulating endocrine signals—hormones like ghrelin, glucagon‐like peptide‐1 and leptin, that have a major impact on the CNS mechanisms controlling food intake and energy balance. We discuss signaling mechanisms that mediate communication between astrocytes and neurons and consider how these mechanisms are recruited by astrocytes activated in response to various metabolic challenges. We review experimental data suggesting that astrocytes modulate the activities of the respiratory and autonomic neuronal networks that ensure adaptive changes in breathing and sympathetic drive in order to support the physiological and behavioral demands of the organism in ever‐changing environmental conditions. Finally, we discuss evidence suggesting that altered astroglial function may contribute to the pathogenesis of disparate neurological, respiratory and cardiovascular disorders such as Rett syndrome and systemic arterial hypertension.

Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca2+ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca2+]i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP3R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP3. At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca2+]i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min−1). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP3R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP3R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca2+ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca2+ signaling, each of them generating oscillations with a specific temporal pattern.

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) 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.

Short-term hypoxia induces a selective death of GABAergic neurons

It is known that brief episodes of hypoxia protect neurons from death caused by global ischemia and hypoxia (hypoxic preconditioning). At the same time, brief hypoxia may cause a phenomenon of posthypoxic hyperexcitability during reoxygenation, which can lead to the death of separate neurons due to their individual differences. In this work we compare the effects of short-term hypoxia on the initiation of preconditioning and posthypoxic hyperexcitability in two populations of neurons: inhibitory GABAergic neurons and excitatory glutamatergic neurons. Preconditioning effect was evaluated according to the suppression of the NMDA-receptor activity. The phenomenon of posthypoxic hyperexcitability was estimated by the appearance of spontaneous synchronized Ca2+ spikes in the neuronal network during reoxygenation after each episode of hypoxia. It is shown that the preconditioning effect occurs only in glutamatergic neurons. In the GABAergic neurons the effect of preconditioning was not observed. The activity of NMDA receptors in these neurons was not suppressed but increased after each episode of hypoxia. At the moment of posthypoxic synchronous Ca2+-spike generation, a global increase of the cytoplasmic Ca2+ concentration occurred in a few of GABAergic neurons, followed by the apoptotic death of these cells. The anti-inflammatory cytokine, interleukin-10 (IL-10) prevented the development of posthypoxic hyperexcitability, inhibiting spontaneous synchronous Ca2+ spike, and protected GABAergic neurons from the death, restoring the preconditioning effect in them. PI3-kinase inhibitors wortmannin and LY294002 prevented the IL-10 protective effect abolishing the inhibiting effect of IL-10 on the generation of the Ca2+ synchronous spike. These findings point out to the leading role of GABAergic neurons in the development of posthypoxic hyperexcitability. We suggest that the reason for posthypoxic hyperexcitability in the network is a weakening of the inhibiting effect of GABAergic neurons. Activation of different signaling pathways leading to activation of PKB- and PKG-dependent phosphorylation in the neurons of this type represents a possible strategy to protect neurons from death during hypoxia.