A. V. Berezhnov

Role of inorganic polyphosphate in mammalian cells: from signal transduction and mitochondrial metabolism to cell death.

Inorganic polyphosphate (polyP) is a polymer compromised of linearly arranged orthophosphate units that are linked through high-energy phosphoanhydride bonds. The chain length of this polymer varies from five to several thousand orthophosphates. PolyP is distributed in the most of the living organisms and plays multiple functions in mammalian cells, it is important for blood coagulation, cancer, calcium precipitation, immune response and many others. Essential role of polyP is shown for mitochondria, from implication into energy metabolism and mitochondrial calcium handling to activation of permeability transition pore (PTP) and cell death. PolyP is a gliotransmitter which transmits the signal in astrocytes via activation of P2Y1 receptors and stimulation of phospholipase C. PolyP-induced calcium signal in astrocytes can be stimulated by different lengths of this polymer but only long chain polyP induces mitochondrial depolarization by inhibition of respiration and opening of the PTP. It leads to induction of astrocytic cell death which can be prevented by inhibition of PTP with cyclosporine A. Thus, medium- and short-length polyP plays role in signal transduction and mitochondrial metabolism of astrocytes and long chain of this polymer can be toxic for the cells.

Intracellular pH Modulates Autophagy and Mitophagy

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

Interaction of misfolded proteins and mitochondria in neurodegenerative disorders

The number of the people affected by neurodegenerative disorders is growing dramatically due to the ageing of population. The major neurodegenerative diseases share some common pathological features including the involvement of mitochondria in the mechanism of pathology and misfolding and the accumulation of abnormally aggregated proteins. Neurotoxicity of aggregated 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.

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.

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.

Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site

Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5μM, 0.2μM and 1μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca(2+) concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca(2+) oscillations and relatively low resting level of intracellular Ca(2+), nicotine triggered Ca(2+)-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [(125)I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6* nAChR types in specific functions of PMNs.

α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson’s disease

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson’s disease.How toxic aggregated forms of α-synuclein lead to neurodegeneration is unclear. Here authors use biophysical and cellular imaging methods to show that specific oligomers of α-synuclein exert effects on mitochondria to induce opening of the permeability transition pore, leading to cell death in Parkinson’s disease.

Polarographic and spectroscopic studies of the effects of fluoroacetate/fluorocitrate on cells and mitochondria

Experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells (EATC) and cardiomyocytes, exposed to fluoroacetate (FA) or fluorocitrate (FC) in vitro. The effects of FA developed at much higher concentrations in comparison with FC and was dependent upon respiratory substrates: with pyruvate, FA induced a slow oxidation of pyridine nucleotides (NAD(P)H) and inhibition of respiration. NAD(P)H oxidation was prevented by incubation of mitochondria with cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore. Studies of the NAD(P)H level and calcium response generated in EATC under activation with ATP via the metabotropic P2Y receptor, revealed a loss of NAD(P)H from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H on exposure to FA. An increase of cytosolic (Ca 2+ ) 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 Ca 2+ waves in cardiomyocytes, and cause the hyper- sensitivity of platelets reported on earlier. Highlighting the reciprocal relationship between intracellular NAD(P)H and calcium balance, we discuss metabolic pathway modulation in the context of development of an effective therapy for FA poisoning.

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca2+]i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca2+. Maximal level of Ca2+, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A2 and/or Ca2+/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca2+]i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca2+-sparks and Ca2+-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca2+-microdomain formation, where the “vicious circle” of Ca2+-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca2+-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na+ and Ca2+ (lipid pore).

Sarcolemmal α2-adrenoceptors control protective cardiomyocyte-delimited sympathoadrenal response

Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca2+ current (ICaL) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced β-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of β- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated ICaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation.