Metoda Lipnik-Štangelj

Ethanol and acetaldehyde disturb TNF-alpha and IL-6 production in cultured astrocytes

Ethanol disturbs astroglial growth and differentiation and causes functional alterations. Furthermore, many signalling molecules produced by astrocytes contribute to these processes. The aim of the present study was to investigate the influence of ethanol and its primary metabolite, acetaldehyde, on TNF-alpha and IL-6 production in a rat cortical astrocyte primary culture. We are the first to report that both ethanol and acetaldehyde can modulate TNF-alpha and IL-6 secretion from cultured astrocytes. Long-term exposure (7 days) to ethanol and acetaldehyde was more toxic than an acute (24 hours) exposure. However, both compounds showed a biphasic, hormestic effect on the IL-6 secretion after the acute as well as the long-term exposure, and the maximum stimulation was reached for 50-mM ethanol and 1-mM acetaldehyde after 7-day exposure. In contrast, both compounds reduced the TNF-alpha secretion, where the effect was concentration-dependent. The catalase inhibitor 2-amino-1,2,4 triazole significantly reduced the ethanol toxicity in the cultured astrocytes after the acute as well as the long-term exposure. In conclusion, both ethanol and acetaldehyde affect the production of IL-6 and TNF-alpha in cultured astrocytes. The effect depends on the concentration of the compounds and the duration of the exposure. Acetaldehyde is a more potent toxin than ethanol, and ethanol’s toxicity in the brain is at least partially due to its primary metabolite, acetaldehyde.

Chronic pain treatment: the influence of tricyclic antidepressants on serotonin release and uptake in mast cells.

The involvement of serotonin (5-HT) in chronic pain mechanisms is established. 5-HT inhibits central painful stimuli, but recent data suggests that 5-HT could also enhance pain stimulus from the periphery, where mast cells play an important role. We aimed in our study to clarify the influence of selected tricyclic antidepressants (TCAs) on mast cell function: secretion, uptake, and reuptake of 5-HT, that could interfere with 5-HT levels and in this way contribute to the generation of pain. As an experimental model, we used isolated rat peritoneal mast cells and incubated them with selected TCAs (clomipramine, amitriptyline, doxepin, and imipramine) under different experimental conditions. 5-HT release, uptake, and reuptake were determined spectrofluorometrically. We showed that TCAs were able to inhibit 5-HT secretion from mast cells, as well as uptake of exogenous 5-HT and reuptake of secreted 5-HT back into mast cells. The effects of TCAs were concentration dependent; higher concentrations of TCAs inhibited the secretion of 5-HT induced by compound 48/80, whereas lower concentrations of TCAs inhibited 5-HT uptake. The most effective TCA was halogenated clomipramine. As TCAs are well introduced in chronic pain treatment, the insight into mechanisms of action is important for an understanding of their effect in various pain conditions.

Histamine and astrocyte function

Astrocytes support the brain through numerous functional interactions in health and disease. The recent advances in our knowledge of astrocyte involvement in various neurological disorders raised up several questions about their role and functioning in the central nervous system. From the evidence discussed in this review, we show that histamine importantly influences the main astrocytic activities such as ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These processes are mediated through at least three histamine receptor subtypes, H1, H2 and H3, expressed on the astrocyte surface. Thus, we recognize histamine as an important player in the modulation of astrocytic functions that deserves further considerations in exploring involvement of astrocytes in neurological disorders.

Staurosporine induces different cell death forms in cultured rat astrocytes

Background. Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Materials and methods. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec- 1). Different forms of a cell death were detected using flow cytometry. Results. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10-7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10-6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Conclusion. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis.

Comparison of Ethanol and Acetaldehyde Toxicity in Rat Astrocytes in Primary Culture

Comparison of Ethanol and Acetaldehyde Toxicity in Rat Astrocytes in Primary Culture This study compared the effects of toxicity of ethanol and its first metabolite acetaldehyde in rat astrocytes through cell viability and cell proliferation. The cells were treated with different concentrations of ethanol in the presence or absence of a catalase inhibitor 2-amino-1,2,4 triazole (AMT) or with different concentrations of acetaldehyde. Cell viability was assessed using the trypan blue test. Cell proliferation was assessed after 24 hours and after seven days of exposure to either ethanol or acetaldehyde. We showed that both ethanol and acetaldehyde decreased cell viability in a dose-dependent manner. In proliferation studies, after seven days of exposure to either ethanol or acetaldehyde, we observed a significant dose-dependent decrease in cell number. The protein content study showed biphasic dose-response curves, after 24 hours and seven days of exposure to either ethanol or acetaldehyde. Co-incubation in the presence of AMT significantly reduced the inhibitory effect of ethanol on cell proliferation. We concluded that long-term exposure of astrocytes to ethanol is more toxic than acute exposure. Acetaldehyde is a much more potent toxin than ethanol, and at least a part of ethanol toxicity is due to ethanols first metabolite acetaldehyde. Primerjava toksičnosti etanola in acetaldehida za podganje astrocite v primarni kulturi V študiji smo primerjali toksičnost etanola in njegovega prvega metabolita acetaldehida za podganje astrocite z določitvijo celične viabilnosti in proliferacije. Celične kulture smo tretirali z različnimi koncentracijami etanola, etanola v prisotnosti inhibitorja katalaze 2-amino-1,2,4 triazol-a (AMT) ali z različnimi koncentracijami acetaldehida. Celično viabilnost smo vrednotili s pomočjo testa s tripanskim modrilom, celično proliferacijo pa s štetjem celic in določitvijo koncentracije proteinov po 24-urni, kot tudi 7-dnevni izpostavljenosti. S študijo smo pokazali, da tako etanol kot tudi acetaldehid v odvisnosti od njune koncentracije zmanjšata celično viabilnost. V študiji proliferacije sta etanol in acetaldehid, v odvisnosti od njunih koncentracij, značilno zmanjšala število celic po 7-dnevni izpostavljenosti. Pri ugotavljanju vsebnosti proteinov smo dobili bifazno krivuljo tako po 24-urni, kot tudi po 7-dnevni izpostavljenosti različnim koncentracijam etanola oziroma acetaldehida. Prisotnost AMT je signifikantno zmanjšala učinek etanola na celično proliferacijo. Zaključimo lahko, da je dolgotrajna izpostavljenost astrocitov etanolu bolj toksična kot akutna. Acetaldehid je močnejši toksin kot etanol in vsaj del toksičnosti etanola je posledica delovanja njegovega prvega metabolita, acetaldehida.

The synergistic effect of histamine and IL-6 on NGF secretion from cultured astrocytes is evoked by histamine stimulation of IL-6 secretion via H1-receptor-PKC-MAPK signalling pathway

Histamine is a potent stimulator of nerve growth factor (NGF) production in both the central nervous system and in the periphery. The main signalling pathway, involved in the synthesis and secretion of NGF, includes activation of histamine H1-receptor, stimulation of Ca -dependent protein kinase C (PKC) and mitogen-activated protein (MAP) kinase [1–3]. Besides its direct stimulation of NGF production, histamine is able to stimulate it also indirectly. This effect of histamine results from its interactions with different cytokines, i.e. IL-1 beta and IL-6 which are also able to stimulate production of NGF [5, 6]. We showed in the past that interaction between histamine and interleukin-6 causes a long term synergism on NGF secretion from cultured astrocytes [5, 6]. It is known that histamine can stimulate IL-6 production and expression of IL-6 receptors on certain types of cells such as human lymphoid, monocytoid and hepatoma cell lines. Both effects are dependent on activation of histamine H1-receptors [7]. Therefore, in the present work we further investigated molecular mechanisms of histamine-IL-6 interaction in the regulation of NGF production, and the role of H1-receptor-PKC-MAPK signalling pathway in this process.

Staurosporine induces apoptosis and necroptosis in cultured rat astrocytes

Apoptosis and necroptosis are highly regulated, interconnected forms of a cell death. The distinction between them is critical, because necroptosis may cause significant cell loss and local inflammation, whereas apoptosis is essential for tissue homeostasis. The same stimulus can induce both apoptosis and necroptosis. Both forms of a cell death were detected in various pathologies, including pathologies in the central nervous system. Astrocytes are a large, heterogeneous cell population in the central nervous system, with many supportive, developmental functions. Although their demise may seriously impair normal functions of the central nervous system, it is still poorly understood. In this study, apoptosis and necroptosis were induced in cultured rat astrocytes by staurosporine. When a low concentration (10−7 M) of staurosporine was applied, a significantly increased proportion of early apoptotic cells was detected after regeneration in a staurosporine free medium. The proportion of necroptotic cells was already increased without regeneration after 3 hours of exposure to staurosporine. When a higher (10−6 M) concentration of staurosporine was applied, further significantly increased necroptosis was detected after regeneration in a staurosporine free medium. Necroptosis was significantly reduced when RIP1 kinase was inhibited by necrostatin-1, whereas inhibition of caspases with z-vad-fmk, an irreversible pan-caspase inhibitor, did not prevent necroptosis. This report of necroptosis induced by staurosporine represents a simple approach for the in vitro induction and detection of apoptosis and necroptosis.

Effects of histamine and IL-1β on PKC-stimulated nerve growth factor secretion from glial cells

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The effect of some anti-inflammatory drugs on histamine and serotonin release from rat peritoneal mast cells

A number of anti-inflammatory drugs, used in the treatment of inflammation and pain due to their inhibitory effect on prostaglandin synthesis, also display non-specific effects on various cells. Rat peritoneal mast cells are an important source of biogenic amines and other inflammatory mediators. Non-steroidal anti-inflammatory drugs have been reported to inhibit, potentiate or have no effect on histamine release [1]. However, there are no equivalent data reporting the effects of these compounds on serotonin release, which we have previously shown to be secreted in different proportions to histamine [2]. In this study, we therefore investigated the effects of some acidic non-steroidal anti-inflammatory drugs (acetylsalicylic acid, diclofenac, indomethacin) and a structurally unrelated drug (auranofin) on the secretion of both amines from rat peritoneal mast cells.

Chapter 8 The Impact of Astrocytes in the Clearance of Neurotransmitters by Uptake and Inactivation

Abstract Astrocytes, which are no longer considered as passive supportive cells of central nervous system, actively participate in brain communication as well as take care for proper microenvironment, because they take up the excess of extracellular potassium ions and neurotransmitters. Neurotransmitter transporters are key elements in terminating synaptic activity of distinct neurotransmitters. They use energy stored in the electrochemical gradient of either sodium ions or protons across neuronal or glial cell membrane for uphill transport of neurotransmitters from extracellular to intracellular site. Sodium‐coupled neurotransmitter transporters belong to two distinct families of transporters of solute carrier proteins (SLC): SLC 1 which represents glutamate transporters and SLC6 to which the other neurotransmitter transporters belong. Apart from neurotransmitter transporters, there is another family in the SLC transporter super family that participates in movement of some monoamines across membranes, a SLC22 family. They act in a sodium‐ and chloride‐independent manner. Because of direct involvement of transporters in the availability of neurotransmitters, they represent a site of action of many present and future drugs. In the present review we would like to address the importance of neurotransmitter transporters on astrocytes in the regulation of synaptic signaling.