Marina Sergeeva

Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A2 and is differently regulated by cyclic AMP and Ca2

Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. Primary astrocytes were cultured under standard conditions and prelabeled with [14C]DHA or with [3H]AA. Adenosine 5′‐triphosphate (ATP) (20 μM applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca2+‐independent phospholipase A2 (iPLA2), since it is blocked by the selective iPLA2 inhibitor 4‐bromoenol lactone (BEL, 5 μM) and is not affected either by removal of Ca2+ from extracellular medium or by suppression of intracellular Ca2+ release through PLC inhibitor (U73122, 5 μM). AA release, on the other hand, which is stimulated by ATP, is attributed to Ca2+‐dependent cytosolic PLA2 (cPLA2). AA release is abolished by U73122 and, by removal of extracellular Ca2+, is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 μM), a general inhibitor of intracellular PLA2 s. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA2 and 40 kDa protein reactive to iPLA2 antibodies. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 μM), and by the protein kinase A (PKA) activator dibutyryl‐cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl‐cAMP, but is almost completely blocked by 2,3‐dideoxyadenosine (20 μM) and inhibited by 34% by H89 (10 μM), inhibitors of adenylyl cyclase and PKA, respectively. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. Different sensitivities of iPLA2 and cPLA2 to Ca2+ and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.

Role of Ca2+-independent phospholipase A2 and n-3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation.

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A2 and cyclooxygenases. These enzymes release the prostaglandin precursor, the n−6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H2. Docosahexaenoic acid, which belongs to the n−3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase‐2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca2+ signaling, which results in changes of activity of Ca2+‐dependent phospholipase A2, hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A2, i.e. Ca2+‐dependent phospholipase A2 and Ca2+‐independent phospholipase A2, respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca2+‐ and cAMP‐dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca2+‐independent phospholipase A2 and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca2+‐independent phospholipase A2 and docosahexaenoic acid might be crucially involved in brain‐specific regulation of prostaglandins.

Prostaglandin synthesis in rat brain astrocytes is under the control of the n‐3 docosahexaenoic acid, released by group VIB calcium‐independent phospholipase A2

In the current study, we reveal that in astrocytes the VIB Ca2+‐independent phospholipase A2 is the enzyme responsible for the release of docosahexaenoic acid (22:6n‐3). After pharmacological inhibition and siRNA silencing of VIB Ca2+‐independent phospholipase A2, docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n‐6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca2+‐independent phospholipase A2 significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase‐1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase‐1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide‐stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca2+‐independent phospholipase A2 negatively controls prostaglandin production.

Peroxisome Proliferator-Activated Receptor (PPAR)-γ Positively Controls and PPARα Negatively Controls Cyclooxygenase-2 Expression in Rat Brain Astrocytes through a Convergence on PPARβ/δ via Mutual Control of PPAR Expression Levels

Peroxisome proliferator-activated receptor (PPAR) transcription factors are pharmaceutical drug targets for treating diabetes, atherosclerosis, and inflammatory degenerative diseases. The possible mechanism of interaction between the three PPAR isotypes (α, β/δ, and γ) is not yet clear. However, this is important both for understanding transcription factor regulation and for the development of new drugs. The present study was designed to compare the effects of combinations of synthetic agonists of PPARα [2-[4-[2-[4-cyclohexylbutyl (cyclohexylcarbamoyl)amino]ethyl]phenyl] sulfanyl-2-methylpropanoic acid (GW7647)], PPARβ/δ [4-(3-(2-propyl-3-hydroxy-4-acetyl)phenoxy)propyloxyphenoxy acetic acid, (L-165041)], and PPARγ (rosiglitazone, ciglitazone) on inflammatory gene regulation in rat primary astrocytes. We measured cyclooxygenase-2 (COX-2) expression and prostaglandin E2 synthesis in lipopolysaccharide (LPS)-stimulated cells. PPARα, PPARβ/δ, and PPARγ knockdown models served to delineate the contribution of each PPAR isotype. Thiazolidinediones enhanced the LPS-induced COX-2 expression via PPARγ-dependent pathway, whereas L-165041 and GW7647 had no influence. However, the addition of L-165041 potentiated the effect of PPARγ activation through PPARβ/δ-dependent mechanism. On the contrary, PPARα activation (GW7647) suppressed the effect of the combined L-165041/rosiglitazone application. The mechanism of the interplay arising from combined applications of PPAR agonists involves changes in PPAR expression levels. A PPARβ/δ overexpression model confirmed that PPARβ/δ expression level is the point at which PPARγ and PPARα pathways converge in control of COX-2 gene expression. Thus, we discovered that in primary astrocytes, PPARγ has a positive influence and PPARα has a negative influence on PPARβ/δ expression and activity. A positive/negative-feedback loop is formed by PPARβ/δ-dependent increase in PPARα expression level. These findings elucidate a novel principle of regulation in the signaling by synthetic PPAR agonists that involves modulating the interaction between PPARα,-β/δ, and -γ isoforms on the level of their expression.

Disruption of actin cytoskeleton in cultured rat astrocytes suppresses ATP- and bradykinin-induced [Ca2+]i oscillations by reducing the coupling efficiency between Ca2+ release, capacitative Ca2+ entry, and store refilling

Oscillations of [Ca(2+)](i) which are believed to be important in regulation of cellular behaviour or gene expression, require Ca(2+) entry via capacitative Ca(2+) influx for store refilling. However, the mediator between Ca(2+) store content and activation of Ca(2+) influx is still elusive. There is also controversy about the role of the actin cytoskeleton in this coupling. Therefore, the importance of an intact actin cytoskeleton on ATP- and bradykinin-elicited Ca(2+) signalling was investigated in cultured rat astrocytes by treatment with cytochalasin D which changes the morphology of the cells from an extended to a rounded shape. Cytochalasin D-treated astrocytes were unable, upon prolonged stimulation with the P2Y receptor agonist ATP, to generate oscillations of [Ca(2+)](i) which are, however, seen in 54% of untreated control cells. In cytochalasin D-treated cells, the amplitude of the initial Ca(2+) response was reduced mainly by disturbing the Ca(2+) influx, and, moreover, the total Ca(2+) pool which is sensitive to thapsigargin or cyclopiazonic acid was diminished.Thus, disruption of the cytoskeleton blocks agonist-elicited [Ca(2+)](i) oscillations apparently by reducing the coupling efficiency between intracellular Ca(2+) stores and capacitative Ca(2+) entry.

Exploring the Transcriptome of Ciliated Cells Using In Silico Dissection of Human Tissues

Cilia are cell organelles that play important roles in cell motility, sensory and developmental functions and are involved in a range of human diseases, known as ciliopathies. Here, we search for novel human genes related to cilia using a strategy that exploits the previously reported tendency of cell type-specific genes to be coexpressed in the transcriptome of complex tissues. Gene coexpression networks were constructed using the noise-resistant WGCNA algorithm in 12 publicly available microarray datasets from human tissues rich in motile cilia: airways, fallopian tubes and brain. A cilia-related coexpression module was detected in 10 out of the 12 datasets. A consensus analysis of this modules gene composition recapitulated 297 known and predicted 74 novel cilia-related genes. 82% of the novel candidates were supported by tissue-specificity expression data from GEO and/or proteomic data from the Human Protein Atlas. The novel findings included a set of genes (DCDC2, DYX1C1, KIAA0319) related to a neurological disease dyslexia suggesting their potential involvement in ciliary functions. Furthermore, we searched for differences in gene composition of the ciliary module between the tissues. A multidrug-and-toxin extrusion transporter MATE2 (SLC47A2) was found as a brain-specific central gene in the ciliary module. We confirm the localization of MATE2 in cilia by immunofluorescence staining using MDCK cells as a model. While MATE2 has previously gained attention as a pharmacologically relevant transporter, its potential relation to cilia is suggested for the first time. Taken together, our large-scale analysis of gene coexpression networks identifies novel genes related to human cell cilia.

Desensitisation of protease‐activated receptor‐1 (PAR‐1) in rat astrocytes: evidence for a novel mechanism for terminating Ca2+ signalling evoked by the tethered ligand

1 Protease‐activated receptor‐1 (PAR‐1), a G‐protein‐coupled receptor, is activated when thrombin cleaves its N‐terminal exodomain, thereby regulating morphology, growth and survival of neurones and astrocytes. We have investigated the mechanism of PAR‐1 desensitisation and resensitisation after proteolytic or non‐proteolytic stimulation with thrombin or thrombin receptor agonist peptide (TRag), respectively. 2 In rat primary astrocytes, short‐term stimulation with thrombin resulted in a single [Ca2+]i transient and a dose‐dependent de‐ and resensitisation, as assessed by single‐cell Ca2+ imaging of fura‐2‐loaded astrocytes. 3 An initial proteolytic activation of astrocyte PAR‐1 by exposure to thrombin strongly decreased the response elicited by subsequent application of a second dose of thrombin or of TRag. In contrast, after an initial non‐proteolytic activation of astrocyte PAR‐1 by TRag, the subsequent response to thrombin, but not to an additional application of TRag, was strongly attenuated, and the time course for desensitisation was slower. 4 Based on this finding we hypothesised that after PAR‐1 activation, the ‘tethered ligand’ is proteolytically destroyed. As a consequence, the receptor becomes unresponsive to a subsequent thrombin stimulus but is still capable of responding to TRag. This hypothesis was supported by applying thermolysin, which is known to cleave PAR‐1 within its tethered‐ligand domain, and was confirmed by incubation with soybean trypsin inhibitor. 5 PAR‐1 resensitisation occurs via new PAR‐1 synthesis since resensitisation was inhibited by cycloheximide and brefeldin A. 6 From these results, we derive a novel model wherein activation of PAR‐1, in addition to initiating signal transduction, activates a protease mechanism that cleaves the N‐terminus of the receptor, thus terminating the signal and probably inducing receptor internalisation.

Arachidonic acid in astrocytes blocks Ca2+ oscillations by inhibiting store-operated Ca2+ entry, and causes delayed Ca2+ influx

ATP-elicited oscillations of the concentration of free intracellular Ca(2+) ([Ca(2+)](i)) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca(2+) store refilling. Short-term application of AA, but not ETYA, blocked Ca(2+) influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca(2+)](i) oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca(2+)](i) increase. This AA-induced [Ca(2+)](i) rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 microM Gd(3+), indicative for the influx of extracellular Ca(2+). Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca(2+)](i), firstly, a rapid reduction of capacitative Ca(2+) entry thereby suppressing [Ca(2+)](i) oscillations, and secondly inducing a delayed activation of Ca(2+) entry, also sensitive to low Gd(3+) concentration.

Prostaglandin E2 biphasic control of lymphocyte proliferation: inhibition by picomolar concentrations

Prostaglandins (PGs) have an important physiological role in the modulation of various cell immune functions. The main sources of PGs during immune responses are monocyte cells. We report here the ability of non‐stimulated macrophages to synthesize prostanoids and show that peritoneal mouse macrophages synthesize PGE2, PGF2a and thromboxane B2, spleen macrophages produce PGE2 and PGF2a, and in a fresh medium this synthesis reaches a constant basal level in a few hours. We studied the kinetics of Con A‐induced proliferation of murine splenocytes under the influence of a wide range of PGE2 concentrations (10−14–10−7 M). The suppressive effect of PGE2 decreased when its concentration was lowered and disappeared at 10−9 M PGE2 (this concentration corresponded to the basal level of non‐stimulated macrophage synthesis of PGE2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE2 caused a suppressive effect comparable with that for 10−8 M PGE2. We also found that PGE2 significantly inhibited cell proliferation when it was added 1 h before the addition of mitogen, as compared with simultaneous mitogen addition. The effect was obtained for both low (10−12 M) and high (10−8 M) PGE2 concentrations. This phenomenon of PGE2 biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.

Arachidonic acid and docosahexaenoic acid suppress thrombin-evoked Ca2+ response in rat astrocytes by endogenous arachidonic acid liberation.

Arachidonic (AA) and docosahexaenoic acid (DHA) are the major polyunsaturated fatty acids (PUFAs) in the brain. However, their influence on intracellular Ca2+ signalling is still widely unknown. In astrocytes, the amplitude of thrombin‐ induced Ca2+ response was time‐dependently diminished by AA and DHA, or by the AA tetraynoic analogue ETYA, but not by eicosapentaenoic acid (EPA). Thrombin‐elicited Ca2+ response was reduced (20–30%) by 1‐min exposure to AA or DHA. Additionally, 1‐min application of AA or DHA together with thrombin in Ca2+‐free medium blocked Ca2+ influx, which followed after readdition of extracellular Ca2+. EPA and ETYA, however, were ineffective. Long‐term treatment of astrocytes with AA and DHA, but not EPA reduced the amplitude of the thrombin‐induced Ca2+ response by up to 80%. AA and DHA caused a comparable decrease in intracellular Ca2+ store content. Only DHA and AA, but not EPA or ETYA, caused liberation of endogenous AA by cytosolic phospholipase A2 (cPLA2). Therefore, we reasoned that the suppression of Ca2+ response to thrombin by AA and DHA could be due to release of endogenous AA. Possible participation of AA metabolites, however, was excluded by the finding that specific inhibitors of the different oxidative metabolic pathways of AA were not able to abrogate the inhibitory AA effect. In addition, thrombin evoked AA release via activation of cPLA2. From our data we propose a novel model of positive/negative‐feed‐back in which agonist‐induced release of AA from membrane phospholipids promotes further AA release and then suppresses agonist‐induced Ca2+ responses.