IRON MODULATES NOREPINEPHRINE EFFECT ON ASTROCYTES

Aims: Astrocyte position between synapses and blood vessels allows them to ful l crucial functions such as regulation of synaptic activity and potassium bu ering. Well positioned in the close vicinity of synaptic cleft astrocytes are considered to be a direct target of norepinephrine (NE). Synaptic activity and neurotransmitter actions can be in uenced by extracellular iron. Here we investigated whether iron interacts with NE and if this interaction can modulate astrocyte response to NE. Methods: To investigate the interaction between iron and norepinephrine we used spectrophotometry approach. Iron e ect on astrocyte response to NE was examined by the whole-cell patch-clamp technique. Membrane currents were recorded from cultured cortical astrocytes prepared from WT rats. Results: Using spectrophotometry we observed that iron interacts with NE which leads to the formation of a stable complex in the 1:1 stoichiometry. We also found that iron bound to NE completely blocks NE-induced increase of large-conductance calcium sensitive potassium current in astrocytes. Conclusions: Astrocyte response to NE is modi ed when this neurotransmitter forms a complex with iron. This implies that NE binding to astrocytic noradrenergic receptors may be prevented by iron. Our ndings point toward compromised astrocyte functions related to the potassium bu ering when NE action is modified by iron.kategorija M3

Penicillamine prevents damaging redox in vitro interactions of bilirubin and copper

Toxic effects of unconjugated bilirubin (BR) in neonatal hyperbilirubinemia have been related to redox and/or coordinate interactions with Cu2+. However, the development and mechanisms of such interactions at physiological pH have not been resolved. This study shows that BR reduces Cu2+ to Cu1+ in 1:1 stoichiometry. Apparently, BR undergoes degradation, i.e. BR and Cu2+ do not form stable complexes. The binding of Cu2+ to inorganic phosphates, liposomal phosphate groups, or to chelating drug penicillamine, impedes redox interactions with BR. Cu1+ undergoes spontaneous oxidation by O2 resulting in hydrogen peroxide accumulation and hydroxyl radical production. In relation to this, copper and BR induced synergistic oxidative/damaging effects on erythrocytes membrane, which were alleviated by penicillamine. The production of reactive oxygen species by BR and copper represents a plausible cause of BR toxic effects and cell damage in hyperbilirubinemia. Further examination of therapeutic potentials of copper chelators in the treatment of severe neonatal hyperbilirubinemia is needed

Ligand and redox - interactions of adrenaline with iron at physiological pH

Adrenaline (Adr) is catecholamine that is released by the sympathetic nervous system and adrenal medulla. It is involved in several physiological functions, including regulation of blood pressure, vasoconstriction, cardiac stimulation, and regulation of the blood glucose levels 1 . Transients of high levels of Adr in the bloodstream have been recognized for a long time as a cause of cardiovascular problems that develop under chronic exposure to psychosocial and physical stress 2,3. A number of studies have found a connection between the excess of Adr, cardiotoxic effects, and oxidative stress, that is irrespective of adrenergic receptors stimulation 2-4. The mechanism behind this involves Adr (coordinate and redox) interactions with iron, which are still not clear. Two main concepts have been proposed - Adr autooxidation and redox interactions with iron, the most abundant transition metal in human plasma 5 . Fe3+ is known to build complexes with catechols 6 , but data on Fe3+ coordinate interactions with Adr at physiological pH are missing. In addition to its (patho)physiological role, Adr is of interest from the aspect of development of catecholamine-rich biopolymers with adhesive properties and metelloorganic frameworks 7,8. The adhesion and other properties materials are based on the cross-linking via coordinate bonds with Fe3+ at pH > 7. Finally, ligands might dramatically alter the redox potential of Fe3+/Fe2+ couple 9 . It has been shown that specific ligands with high affinity for Fe3+, including some catechols, might promote the oxidation and increase the reactivity of Fe2+ with molecular oxygen 10. The aim of our study was to examine the nature of Adr interactions with Fe3+ and Fe2+: stoichiometry, sites of coordinate bonds formation and structure of complex(es), and redox activity, at pH 7.4 and different concentration ratios. The coordinate and redox interactions were investigated using UV/Vis spectrophotometry, low temperature EPR, Raman 143 spectroscopy, cyclic voltammetry, and oximetry. The stability of Adr in the studied reactions was monitored by HPLC. At pH 7.4, Adr forms complexes with Fe3+, in the 1:1, and 3:1 stoichiometry, depending on (high or low) Adr/Fe3+ concentration ratio. The high-spin Fe3+ 1:1 and 3:1 complexes show different symmetries, with the 3:1 complex displaying higher EPR spectral anisotropy. Raman spectroscopy showed that oxygen atoms on the catechol ring represent the sites of coordinate bond formation in the bidentate Adr-Fe3+ complex. The bonds appear to be stronger in the 1:1 complex, and not to share the same plane with the ring. On the other hand, Adr and Fe2+ build a complex that acts as a strong reducing agent. In the presence of O2, this leads to the production of H2O2, and to a facilitated formation of Adr/Fe3+ complexes. Adr is not oxidized in this process, i.e. iron is not an electron shuttle but electron donor. Catalyzed oxidation of Fe2+ in the presence of Adr represents a plausible chemical basis of stress-related damage of heart cells. In addition, our results imply that the application/pre-binding of Fe2+ followed by oxidation at pH > 7 might be a simple alternative strategy for promotion of cross-linking in catecholamine-rich biopolymers frameworks

ASTROCYTE ACTIVITY IN THE CENTRAL NERVOUS SYSTEM AUTOIMMUNITY

Aims: Multiple sclerosis (MS) is an in ammatory autoimmune disorder of the central nervous system (CNS). Complex interactions between inltrating immune cells (IIC) and resident glial cells of the CNS cause myelin loss and neuronal dysfunction in MS. Here we aim to understand how naïve astrocytes functionally respond to the IIC invasion of the CNS. Methods: We measured calcium activity of naïve astrocytes in culture upon application of IIC. An experimental autoimmune encephalomyelitis (EAE) MS rat model was used to isolate IIC from the spinal cord of animals at the symptomatic stage. Naïve astrocytes were isolated from the spinal cord of WT rats. Results: We show that IIC and not the lymph node immune cells evoke vigorous increase in the astrocyte calcium activity. This IIC-induced calcium response depends on an autocrine activation of the purinergic P2X7 receptors on the naïve astrocytes.We also show that IIC induce ATP release from astrocytes by a mechanism that involves gap junctions and/or hemichannels activation and not the vesicular pathway. Our data indicate that ATP release and subsequent increase in the astrocytic calcium activity mainly depends on the cell-cell contact between naïve astrocytes and IIC. Conclusions: These results show that naïve astrocytes functionally respond to the IIC by augmented release of ATP. An increase in ATP release would alter astrocyte-neuron communication and a ect neuronal function in MS.kategorija M3

Iron modulates norepinephrine effect on astrocytes

Iron, an essential element for living organisms, participates in a wide range of metabolic processes. It appears predominantly firmly bound to proteins, but can also be loosely bound to low-affinity ligands, referred as labile iron pool (LIP). The composition and amount of LIP can vary considerably under different physiological conditions, playing a beneficial role in iron economy and homeostasis or contributing to the generation of reactive oxygen species. It is still not known if bioactivity of low-affinity ligands can be modulated by iron binding. Catecholamine neurotransmitters including norepinephrine (NE) can chelate iron. In the close vicinity of synaptic cleft, astrocytes are direct target of norepinephrine. Here we show on cultured rat cortical astrocytes that iron bound to NE completely blocks neurotransmitter activity of NE. However, how astrocyte activity changes when norepinephrine binds iron remains unknown. We show, using spectrophotometry that NE and Fe3+ form complex in the 1:1 stoichiometry, at pH 7.4. Iron effect on astrocyte response to NE was examined by the whole-cell patch-clamp technique. NE alone evokes changes in the membrane currents of astrocytes, but such effects were not observed for the NE- Fe3+ complex. Our results demonstrating that iron in the complex with norepinephrine inhibits alpha-adrenergic receptors and modulates astrocyte activity, imply a novel neuromodulatory role for LIP.kategorija M3

Background norepinephrine impacts activity of cortical astrocytes

The neurotransmitter norepinephrine (NE) plays a central role in regulating arousal, attention, cognitive function and stress responses. Unlike fast neurotransmitters which act at synapses, NE is released in the neuropil and performs multiple targeting in the surrounding area. The glial cells astrocytes are a direct target of NE, as they express all adrenergic receptor subtypes and respond with Ca2+ increases to NE. Astroglial responses elicited by strong and transient increases of NE in the brain are well studied, but the effect of a low background NE concentration on astrocytes is unknown. This background level of NE is maintained by basal noradrenergic activity and is constantly present in the brain. Therefore, the response of astrocytes to the background NE could have been unintentionally evoked in previous studies but its effect overlooked. To assess action of background NE on astrocytes we combined the whole-cell patch clamp, immunohistochemistry, Ca2+ imaging and pharmacology. We used cultured cortical astrocytes to bypass NE targeting of multiple cell types. We show that cortical astrocytes detect and respond to the background NE concentration with an increase in intracellular Ca2+. This Ca2+ liberated from intracellular stores further increased large-conductance, Ca2+-sensitive potassium (BK) currents in astrocytes. Notably, immunohistochemistry data showed that BK channels and alpha 1 adrenoreceptor are highly expressed in astrocytes in the rat cortex. Furthermore, stimulation of astrocytes by background NE was inhibited by alpha-adrenoceptor antagonist. Our results suggest that astrocytes maintain basal brain activity by perceiving and responding to the background NE.kategorija M6

Triple perovskite-based triboelectric nanogenerator: a facile method of energy harvesting and self-powered information generator

A modified aqueous sol-gel reaction is used to synthesize a triple perovskite with a composition Sr3Co2WO9 (SCWO). The structural analysis conducted at room temperature reveals the presence of the cubic phase with space group Fm-3m. The microstructure of the as-synthesized SCWO particles shows a different size of particles depicting a polycrystalline nature. Triboelectrification is a trending and unique concept in energy scavenging methodologies with flexibility in choosing from a variety of materials. This paves the way to evolve eco-friendly triboelectric energy harvesters as a replacement for the limitation of batteries. The outstanding dielectric properties and low loss make triple perovskite a promising candidate for TENG. A triboelectric nanogenerator (TP-TENG) was designed, which operates in vertical contact separation mode generating an electrical response of voltage and current of 300 V and 2.2 μA, respectively, for 10 wt% polydimethylsiloxane-Sr3Co2WO9 (PDMS-SCWO) composite film. The power density of the rough surface TP-TENG was 30.5 μW/cm2, which is much higher than the power density of 5.5 μW/cm2 of plain surface TP-TENG. The positive and negative triboelectric layers of the TP-TENG were made up of aluminum and PDMS-SCWO composite film, respectively. Furthermore, the excellent flexibility and durability of TP-TENG make it suitable for sensing various gaits and information signaling (Morse code) in real-time applications, as well as for charging commercial capacitors. © 2021 Elsevier Ltd1

Approach for patch-clamping using an upright microscope with z-axis movable stage

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping

Photo-redox reactions of indole and ferric iron in water

Iron-organic interactions are involved in a variety of environmental phenomena, including photo-redox reactions, iron cycling and bioavailability, as well as contaminant fate. In this study we examined UV-induced redox reactions of iron and indole in water. The presence of one indole in the irradiated system resulted in the presence of eight reduced ferric ions, not counting direct photolysis of Fe3+ complexes with OH-, which gives Fe2+ and hydroxyl radical (HO center dot) as products. The main mechanisms that contribute to indole-related Fe3+ reduction i.e. Fe2+ accumulation are: (i) HO center dot scavenging, which prevents oxidation of Fe2+ by HO center dot; (ii) oxidation of indole and its derivatives by excited ferric iron; (iii) reduction of ferric iron by excited indole (not present under UV-A). Hydrated electrons released by UV-B-excited indole play only a minor role in the reduction of iron. Indole-derived radicals emerged as byproducts of indole/iron photochemistry. H-1 NMR and low-T EPR spectroscopy showed that indole forms a weak low-symmetry complex with Fe3+. The strongest interactions between iron and pi-cloud in the indole ring are at positions 2, 3, and 7. The formation of complex promotes electron transfer from excited indole to Fe3+. Our findings are important for understanding the catalysis of photo-reduction of iron by heterocyclic aromatic pollutants, and for the development of protocols for indole processing in wastewaters