Fluoxetine affects GluK2 editing, glutamate-evoked Ca(2+) influx and extracellular signal-regulated kinase phosphorylation in mouse astrocytes.

BACKGROUND: We sought to study the effects of chronic exposure to fluoxetine - a selective serotonin reuptake inhibitor (SSRI) and specific 5-HT(2B) receptor agonist in astrocytes - on the expression of kainate receptors (GluK1-5) in cultured astrocytes and in intact brains in mice and on GluK2 editing by adenosine deaminase acting on RNA (ADAR), as well as the ensuing effects of fluoxetine on glutamate-mediated Ca(2+) influx and extracellular signal-regulated kinase (ERK)(1/2) phosphorylation in astrocytes. METHODS: We performed reverse transcription-polymerase chain reaction (PCR) to assess mRNA expression. We analyzed RNA editing with amplification refractory mutation system PCR and complementary DNA sequencing. Protein expression and ERK phosphorylation were assessed using Western blots. We studied gene silencing with specific small interfering RNAs (siRNA), and we studied intracellular Ca(2+) using fluorometry. RESULTS: All GluK subunits were present in the brain in vivo, and GluK2-5 subunits were present in cultured astrocytes. Fluoxetine upregulated GluK2 and ADAR2. Enhanced GluK2 editing by fluoxetine abolished glutamate-mediated increases in intra cellular Ca(2+) and ERK(1/2) phosphorylation. Enhanced editing of GluK2 was prevented by siRNA against the 5-HT(2B) receptor or ADAR2. LIMITATIONS: Limitations of our study include the use of an in vitro system, but our cultured cells in many respects behave like in vivo astrocytes. CONCLUSION: Fluoxetine alters astrocytic glutamatergic function.

Fluoxetine-mediated 5-HT2B receptor stimulation in astrocytes causes EGF receptor transactivation and ERK phosphorylation.

RATIONALE: Fluoxetine has relatively high affinity for Gq/11 protein-coupled 5-HT(2) receptors. Part of these receptors in brain are on astrocytes, where fluoxetine causes an increase in free cytosolic calcium concentration ([Ca(2+)](i)) and phosphorylation of extracellular regulated kinase 1 and 2 (ERK(1/2)). OBJECTIVE: The objectives of the study are to identify subtype of the 5-HT(2) receptor involved, to establish whether ERK(1/2) phosphorylation is a result of 5-HT(2)-mediated transactivation of epidermal growth factor (EGF) receptors (EGFRs), and to determine signaling pathways up- and downstream of ERK(1/2). MATERIALS AND METHODS: Primary cultures of mouse astrocytes, which express all three subtypes of the 5-HT(2) receptor but no 5-HT(2) transporter, were used. ERK(1/2) phosphorylation and c-Fos and FosB protein expression were determined with Western blotting, and c-fos and fosB mRNA expression with reverse transcription polymerase chain reaction. Receptor subtype was investigated with subtype-specific 5-HT antagonists and 5-HT(2B) receptor depletion and signaling pathways by EGFR phosphorylation, using immunoprecipitation and Western blotting, inhibition of protein kinase C (PKC), and [Ca(2+)](i) chelation by BAPTA/AM. RESULTS: ERK(1/2) phosphorylation was abolished by SB204741, a universal 5-HT(2) receptor antagonist, and in 5-HT(2B) receptor-depleted cells, but unaffected by 5-HT(2A) or 5-HT(2C) receptor antagonists (M100907 and SB242084). Phosphorylation of ERK(1/2) and EGFRs was abolished by AG 1478, an inhibitor of EGFR tyrosine kinases, and GM 6001, an inhibitor of Zn-dependent metalloproteinases, suggesting growth factor "shedding" and transactivation of EGFRs. Chelation of [Ca(2+)](i) or PKC inhibition with GF 109203X abrogated ERK(1/2) phosphorylation. Up-regulated mRNA and protein expression of c-fos and fosB was abolished by SB204741, AG1478, and by U0126, an inhibitor of ERK phosphorylation by MAP kinase/ERK kinase.

Ouabain binding kinetics and FXYD7 expression in astrocytes and neurons in primary cultures: implications for cellular contributions to extracellular K+ homeostasis?

Although Na+,K+-ATPase-mediated K+ uptake into astrocytes plays a major role in re-establishing resting extracellular K+ following neuronal excitation little information is available about astrocytic Na+,K+-ATPase function, let alone mechanisms returning K+ to neurons. The catalytic units of the Na+,K+-ATPase are the astrocyte-specific α2, the neuron-specific α3 and the ubiquitously expressed α1. In the present work, Bmax and KD values for α1, α2 and α3 subunits were computed in cultured cerebro-cortical mouse astrocytes and cerebellar granule neurons by non-linear regression as high-affinity (α2, α3) and low-affinity (α1) [3H]ouabain binding sites, which stoichiometrically equal transporter sites. Cellular expression was also determined of the brain- and α1-ß1 isoform-specific FDYX7, regulating Na+,K+-ATPase efficiency and K+-sensitivity. From ouabain-sensitive K+ uptake rates published by ourselves (Walz and Hertz, 1982) or others (Atterwill et al., 1985), Na+,K+-ATPase turnover was determined. Subunits α2 and α3 showed Bmax of 15-30 pmol/mg protein, with maximum turnover rates of 70-80/s. Bmax of the α1 subunit was low in neurons but very high in astrocytes (645 pmol/mg protein), where turnover rate was slow, reflecting expression of selectively expressed FXYD7, and binding was increased by K+. The role of these characteristics for K+ homeostasis are discussed.

Serotonin increases ERK1/2 phosphorylation in astrocytes by stimulation of 5-HT2B and 5-HT2C receptors.

We have previously shown that fluoxetine causes ERK(1/2) phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT(2B) receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK(1/2) phosphorylation by stimulation of 5-HT(2B) receptors with high affinity (EC(50): 20-30 pM), and by stimulation of 5-HT(2C) receptor with low affinity (EC(50): 1 microM or higher). ERK(1/2) phosphorylation induced by stimulation of either 5-HT(2B) or 5-HT(2C) receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT(2B) receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT(2B)-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT(2B) receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT(2B) receptors. In contrast the relevance of the observed 5-HT(2C) receptors on astrocytes is questioned.

Astrocytic transactivation by alpha2A-adrenergic and 5-HT2B serotonergic signaling.

EGF receptor transactivation has been known for more than ten years. It is a signal pathway in which a G-protein-coupled receptor (GPCR) signal leads to release of a growth factor, which in turn activates the EGF receptor-tyrosine kinase in the same or adjacent cells. Astrocytes express a number of GPCRs and play key roles in brain function. Astrocytic transactivation is of special interest, since its autocrine effect may regulate gene expression and alter cell functions in the cells themselves and its paracrine effect may provide additional opportunities for cross-talk between astrocytes and their neighbors, such as neurons. The signal pathways of EGF transactivation are complicated. This does not only apply to the pathways leading to shedding of growth factor(s), but also to the downstream signal pathways of the EGF receptor, i.e., MAPK and PI3K. The latter may vary according to the type of growth factor released, the sites of tyrosine phosphorylation on the EGF receptor, and the duration of the phosphorylation. Using primary cell cultures we have found that dexmedetomidine, a specific alpha(2)-adrenergic receptor, induced shedding of HB-EGF from astrocytes, which in turn transactivated EGF receptors and stimulated astrocytic c-Fos and FosB expression. At the same time released HB-EGF protected neurons from injury caused by H(2)O(2). We have also confirmed dexmedetomidine transactivation in the brain in vivo. EGF transactivation by 5-HT(2B) receptor stimulation was responsible for up-regulation of cPLA(2) in astrocytes by fluoxetine, an antidepressant and inhibitor of the serotonin transporter, which also is a specific 5-HT(2B) agonist.

5-HT2B receptors are expressed on astrocytes from brain and in culture and are a chronic target for all five conventional 'serotonin-specific reuptake inhibitors'.

In well-differentiated primary cultures of mouse astrocytes, which express no serotonin transporter (SERT), the 'serotonin-specific reuptake inhibitor' (SSRI) fluoxetine leads acutely to 5-HT2B receptor-mediated, transactivation-dependent phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) with an EC50 of ~5 µM, and chronically to ERK1/2 phosphorylation-dependent upregulation of mRNA and protein expression of calcium-dependent phospholipase A2 (cPLA2) with ten-fold higher affinity. This affinity is high enough that fluoxetine given therapeutically may activate astrocytic 5-HT2B receptors (Li et al., 2008, 2009). We now confirm the expression of 5-HT2B receptors in astrocytes freshly dissociated from mouse brain and isolated by fluorescence-activated cell sorting (FACS) and investigate in cultured cells if the effects of fluoxetine are shared by all five conventional SSRIs with sufficiently high affinity to be relevant for mechanism(s) of action of SSRIs. Phosphorylated and total ERK1/2 and mRNA and protein expression of cPLA2a were determined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Paroxetine, which differs widely from fluoxetine in affinity for SERT and for another 5-HT2 receptor, the 5-HT2C receptor, acted acutely and chronically like fluoxetine. One micromolar of paroxetine, fluvoxamine or sertraline increased cPLA2a expression during chronic treatment; citalopram had a similar effect at 0.1-0.5 µM; these are therapeutically relevant concentrations.

Down-regulation of GluK2 kainate receptor expression by chronic treatment with mood-stabilizing anti-convulsants or lithium in cultured astrocytes and brain, but not in neurons.

Recent studies have indicated that glutamatergic transmission may be altered in bipolar disorder and affected by chronic treatment with mood-stabilizing drugs. Kainate receptors may be of special interest because i) they have a modulatory role in synaptic transmission, long-term potentiation (LTP) and long-term depression (LDP); and ii) involvement of the kainate receptor subunit GluK2 (GluR6) in behavioral symptoms thought characteristic of mania has been demonstrated in knock-out mice. Glutamate receptors are expressed not only on neurons, but also on astrocytes, where they contribute to regulation of synaptic activity. We have previously shown that primary cultures of mouse astrocytes respond to chronic but not acute treatment with therapeutic relevant concentrations of any of the 'classical' mood-stabilizing drugs, lithium ion (Li(+)), carbamazepine or valproate, with changes in uptake of myo-inositol, cPLA(2) expression and intracellular pH. In the present work, we found i) similar gene expression of the GluK2 subunit of the kainate receptor family in primary cultures of mouse astrocytes and in brain in vivo; ii) a reduction of mRNA and protein expression of GluK2 in astrocytes and in brain after chronic treatment with carbamazepine but no effect in neurons; iii) similar down-regulation in astrocytes by oxcarbamazepine, valproic acid or Li(+), which all have mood-stabilizing effect, but not by the anti-convulsant topiramate, which has no such activity; and iv) abrogation of a normally occurring glutamate-induced ERK phosphorylation in the cultured astrocytes after chronic treatment with any of the mood-stabilizing drugs mentioned above. Possible relationships between these and previously demonstrated effects are discussed.

Chronic treatment of astrocytes with therapeutically relevant fluoxetine concentrations enhances cPLA2 expression secondary to 5-HT2B-induced, transactivation-mediated ERK1/2 phosphorylation.

INTRODUCTION: We have recently shown that fluoxetine, a serotonin-specific reuptake inhibitor (SSRI), has low micromolar affinity for the 5-HT(2C) receptor (but not for 5-HT(2A) and 5-HT(2B) receptors) in primary cultures of mouse astrocytes. This was determined as phosphorylation (stimulation) of extracellular-regulated kinase 1 and 2 (ERK(1/2)) by transactivation-mediated phosphorylation of the epidermal growth factor (EGF) receptor, followed by conventional EGF receptor signaling (Li et al., Psychopharmacology 194:333-334, 2007). Paroxetine has an identical effect. The present study shows that chronic fluoxetine treatment with even higher affinity (EC(50) = 0.5-2.0 microM) upregulates Ca(2+)-dependent phospholipase A(2) (cPLA(2)), which releases arachidonic acid from the sn-2 position of membrane-bound phospholipid, without effect on secretory PLA(2) (sPLA(2)) and intracellular PLA(2) (iPLA(2)). DISCUSSION: This demonstration replicates the fluoxetine-induced cPLA(2) upregulation in rat brain shown by Rao et al. (Pharmacogenomics J 6:413-420, 2006) and provides the new information that upregulation (1) occurs in astrocytes, (2) is evoked by stimulation of 5-HT(2B) receptor, and (3) requires transactivation-mediated ERK(1/2) phosphorylation. Similar upregulation of cPLA(2) in intact brain in response to 5-HT(2)-mediated signaling by elevated serotonin levels and/or an SSRI during antidepressant treatment may explain the repeatedly reported ability of SSRIs to normalize regional decreases which occur in brain metabolism during major depression, since (1) arachidonic acid strongly stimulates glucose metabolism in cultured astrocytes (Yu et al., J Neurosci Res 64:295-303, 1993) and (2) plasma concentrations of arachidonic acid in depressed patients are linearly correlated with regional brain glucose metabolism (Elizabeth Sublette et al., Prostaglandins Leukot Essent Fatty Acids 80:57-64, 2009).