P2Y12 receptor-mediated cyclooxygenase 2 (COX-2) expression enhances tumor cell progression in a mouse model of lymphoma.

The pro-inflammatory enzyme cyclooxygenase 2 (COX-2) has been known to impart metastatic property to cancer cells. However, blocking of COX-2 with nonsteroidal anti-inflammatory drugs or COX-2-specific inhibitors has failed in clinical trials due to adverse effects associated with their prolonged use. We have previously shown that extracellular ATP (eATP), a major component of the tumor microenvironment, enhances COX-2 expression several-fold, both in macrophages and in various cancer cells, by acting on purinergic (P2) receptors. In this study, we show that blocking of P2 receptors significantly reduced tumor growth in a mouse model of lymphoma. Tumors were induced in mice through subcutaneous injection of syngeneic EL4 lymphoma cells. Various P2 receptor antagonists were injected within the tumors after they were palpable. The broad-spectrum P2 receptor antagonist, suramin, P2X7 receptor-specific antagonist, oATP, P2Y6 receptor-specific antagonist, MRS 2578, and P2Y12 receptor-specific antagonist, AR-C 69931, all showed significant arrest in tumor growth. Both suramin and AR-C 69931-treated tumors showed strong reduction in COX-2 expression and modulation of various metastatic markers. Disaggregated cells from AR-C 69931-treated tumors, when injected intravenously in naïve mice, did not exhibit metastasis in various tissues which was observed in mice injected with cells from saline-treated tumors. Our results show that blocking of P2 receptors is a therapeutic alternative to inhibit COX-2 expression, and thereby, arrest tumor progression and metastasis.

Imidazopyridazine Acetylcholinesterase Inhibitors Display Potent Anti-Proliferative Effects in the Human Neuroblastoma Cell-Line, IMR-32.

Imidazo[1,2-b]pyridazine compounds are a new class of promising lead molecules to which we have incorporated polar nitro and amino moieties to increase the scope of their biological activity. Two of these substituted 3-nitro-6-amino-imidazo[1,2-b]pyridazine compounds (5c and 5h) showed potent acetylcholinesterase (AChE) inhibitory activity (IC50 40-50 nM), which we have previously reported. In this study, we wanted to test the biological efficacy of these compounds. Cytotoxicity assays showed that compound 5h mediated greater cell death with over 43% of cells dead at 100 µM and activation of caspase 3-mediated apoptosis. On the other hand, compound 5c mediated a dose-dependent decrease in cell proliferation. Both compounds showed cell cycle arrest in the G0/G1 phase and reduced cellular ATP levels leading to activation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced mitochondrial oxidative stress. It has to be noted that all these effects were observed at doses beyond 10 µM, 200-fold above the IC50 for AChE inhibition. Both compounds also inhibited bacterial lipopolysaccharide-mediated cyclooxygenase-2 and nitric oxide release in primary rat microglial cells. These results suggested that the substituted imidazo (1,2-b) pyridazine compounds, which have potent AChE inhibitory activity, were also capable of antiproliferative, anti-migratory, and anti-inflammatory effects at higher doses.

Mutant Ataxin-3-Containing Aggregates (MATAGGs) in Spinocerebellar Ataxia Type 3: Dynamics of the Disorder.

Spinocerebellar ataxia type 3 (SCA3) is the most common type of SCA worldwide caused by abnormal polyglutamine expansion in the coding region of the ataxin-3 gene. Ataxin-3 is a multi-faceted protein involved in various cellular processes such as deubiquitination, cytoskeletal organisation, and transcriptional regulation. The presence of an expanded poly(Q) stretch leads to altered processing and misfolding of the protein culminating in the production of insoluble protein aggregates in the cell. Various post-translational modifications affect ataxin-3 fibrillation and aggregation. This review provides an exhaustive assessment of the various pathogenic mechanisms undertaken by the mutant ataxin-3-containing aggregates (MATAGGs) for disease induction and neurodegeneration. This includes in-depth discussion on MATAGG dynamics including their formation, role in neuronal pathogenesis, and the debate over the toxic v/s protective nature of the MATAGGs in disease progression. Additionally, the currently available therapeutic strategies against SCA3 have been reviewed. The shift in the focus of such strategies, from targeting the steps that lead to or reduce aggregate formation to targeting the expression of mutant ataxin-3 itself via RNA-based therapeutics, has also been presented. We also discuss the intriguing promise that various growth and neurotrophic factors, especially the insulin pathway, hold in the modulation of SCA3 progression. These emerging areas show the newer directions through which SCA3 can be targeted including various preclinical and clinical trials. All these advances made in the last three decades since the discovery of the ataxin-3 gene have been critically reviewed here.

Exogenous ATP modulates PGE2 release in macrophages through sustained phosphorylation of CDK9 and p38 MAPK.

An important mediator of inflammation is prostaglandin E2 (PGE2 ), whose levels are determined by the activity of the enzyme cyclooxygenase (COX). Of the two isoforms of the enzyme, COX-2 has been shown to be induced in macrophages during inflammation. Although general COX inhibitors, belonging to the class of nonsteroidal anti-inflammatory drugs, or specific COX-2 inhibitors, called coxibs, are useful in the control of acute inflammation, adverse reactions were seen when used chronically in the treatment of rheumatoid arthritis or neurodegenerative diseases. Extracellular ATP (eATP) has been reported as a damage-associated molecular pattern signal. In this report, we show that eATP synergistically increases the levels of COX-2 enzyme and PGE2 in LPS-activated RAW264.7 macrophages and human monocytes. Activation of macrophages also occurred when cultured in media obtained from dying neurons that contained higher levels of ATP. We show that eATP increases the levels of COX-2 protein, which is sustained up to 36 h poststimulation. This is in turn due to sustained levels of phosphorylated, or activated, cyclin-dependent kinase 9 and p38 MAPK in ATP-treated cells compared to LPS-stimulated cells. The eATP-dependent increase in COX-2/PGE2 levels in LPS-activated RAW264.7 cells could be abolished using antagonists for purinergic P2X7 -and P2Y6 receptors. Similarly, the increase in COX-2/PGE2 levels in the peritoneum of LPS-treated mice could be significantly abolished in mice that were preinjected with the nonspecific P2 receptor antagonist, suramin. P2 receptor antagonists, therefore, should be explored in our search for an ideal anti-inflammatory candidate.

Extracellular ATP Mediates Cancer Cell Migration and Invasion Through Increased Expression of Cyclooxygenase 2.

The tumor microenvironment plays a major role in the ability of the tumor cells to undergo metastasis. A major player of tumors gaining metastatic property is the inflammatory protein, cyclooxygenase 2 (COX-2). Several tumors show upregulation of this protein, which has been implicated in mediating metastasis in various cancer types such as of colon, breast and lung. In this report, we show that the concentration of extracellular ATP (eATP) is increased in response to cell death mediated by chemotherapeutic agents such as doxorubicin. By using three different cell-lines-HeLa (cervical), IMR-32 (neuronal) and MCF-7 (breast)-we show that this eATP goes on to act on purinergic (P2) receptors. Among the various P2 receptors expressed in these cells we identified P2X7, in IMR-32 and MCF-7 cells, and P2Y12, in HeLa cells, as important in modulating cell migration and invasion. Downstream of the P2 receptor activation, both p42/44 mitogen-activated protein kinase (MAPK) and the p38 MAPK are activated in these cells. These result in an increase in the expression of COX-2 mRNA and protein. We also observe an increase in the activity of matrix metalloproteinase 2 (MMP-2) enzyme in these cells. Blocking the P2 receptors not only blocks migration and invasion, but also COX-2 synthesis and MMP-2 activity. Our results show the link between purinergic receptors and COX-2 expression. Increased levels of ATP in the tumor microenvironment, therefore, leads to increased COX-2 expression, which, in turn, affords migratory and invasive properties to the tumor. This provides P2 receptor-based anti-inflammatory drugs (PBAIDs) a potential opportunity to be explored as cancer therapeutics.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) Alters Cell Cycle Progression in Human Neuroblastoma Cell Lines.

1-Trichloromethyl-1,2,3,4-tetrahydro-ß-carboline, abbreviated as TaClo, is an endogenous neurotoxin capable of formation in the brain through the condensation of neuronal tryptamine with ingested exogenous toxins such as trichloroethylene or chloral hydrate. Due to its structural resemblance to 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), and similar ability to inhibit mitochondrial complex I, TaClo has been implicated in the etiology of Parkinson's disease. Previous studies have shown the cytotoxicity of TaClo in various cell culture models. In this study, we were interested in identifying the early molecular events within the cell upon exposure to TaClo, a potent mitochondrial toxin. We found increased phosphorylation of 5'-adenosine monophosphate-activated protein kinase (AMPK), induction of autophagy, and a dependence on glycolysis as some of the downstream events to TaClo treatment. Furthermore, TaClo-treated cells undergo accelerated late proliferation but form daughter cells containing fewer neurites, leading to their eventual apoptosis. We also found that TaClo inhibits neuronal prostaglandin E2 synthesis which may play an important role in synaptic plasticity. These results show that TaClo-mediated inhibition of mitochondrial complex I have multiple effects on cellular physiology which are in line with other mitochondrial effectors of Parkinson's disease.

Amyloid beta peptide (25-35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes.

Prostaglandins (PGs) are generated by the enzymatic activity of cyclooxygenase-1 and -2 (COX-1/2) and modulate several functions in the CNS such as the generation of fever, the sleep/wake cycle, and the perception of pain. Moreover, the induction of COX-2 and the generation of PGs has been linked to neuroinflammatory aspects of Alzheimer's disease (AD). Non-steroidal anti-inflammatory drugs (NSAIDs) that block COX enzymatic activity have been shown to reduce the incidence of AD in various epidemiological studies. While several reports investigated the expression of COX-2 in neurons and microglia, expression of COX-2 in astroglial cells has not been investigated in detail. Here we show that amyloid beta peptide 25-35 (Abeta(25-35)) induces COX-2 mRNA and protein synthesis and a subsequent release of prostaglandin E(2) (PGE(2)) in primary midbrain astrocytes. We further demonstrate that protein kinase C (PKC) is involved in Abeta(25-35)-induced COX-2/PGE(2) synthesis. PKC-inhibitors prevent Abeta(25-35)-induced COX-2 and PGE(2) synthesis. Furthermore Abeta(25-35) rapidly induces the phosphorylation and enzymatic activation of PKC in primary rat midbrain glial cells and in primary human astrocytes from post mortem tissue. Our data suggest that the PKC isoforms alpha and/or beta are most probably involved in Abeta(25-35)-induced expression of COX-2 in midbrain astrocytes. The potential role of astroglial cells in the phagocytosis of amyloid and the involvement of PGs in this process suggests that a modulation of PGs synthesis may be a putative target in the prevention of amyloid deposition.

Regional distribution of the prostaglandin E2 receptor EP1 in the rat brain: accumulation in Purkinje cells of the cerebellum.

Prostaglandin E2 (PGE2), is a major prostanoid produced by the activity of cyclooxygenases (COX) in response to various physiological and pathological stimuli. PGE2 exerts its effects by activating four specific E-type prostanoid receptors (EP1, EP2, EP3, and EP4). In the present study, we analyzed the expression of the PGE2 receptor EP1 (mRNA and protein) in different regions of the adult rat brain (hippocampus, hypothalamus, striatum, prefrontal cerebral cortex, parietal cortex, brain stem, and cerebellum) using reverse transcription- polymerase chain reaction, Western blotting, and immunohistochemical methods. On a regional basis, levels of EP1 mRNA were the highest in parietal cortex and cerebellum. At the protein level, we found very strong expression of EP1 in cerebellum, as revealed by Western blotting experiments. Furthermore, the present study provides for the first time evidence that the EP1 receptor is highly expressed in the cerebellum, where the Purkinje cells displayed very high immunolabeling of their perikaryon and dendrites, as observed in the immunohistochemical analysis. Results from the present study indicate that the EP1 prostanoid receptor is expressed in specific neuronal populations, which possibly determine the region-specific response to PGE2.

1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) induces apoptosis in human neuroblastoma cell lines.

The former popular hypnotic chloral hydrate has been known to spontaneously condense with tryptamine in the body to give rise to a highly unpolar 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) derivative. Earlier studies have revealed the relative permeability of the molecule through the body, and its ability to induce Parkinson-like symptoms in rats. In this study, we report that TaClo is a highly cytotoxic agent that leads to apoptosis in human neuroblastoma cells involving caspase-3 activation.

The two-hit hypothesis for neuroinflammation: role of exogenous ATP in modulating inflammation in the brain.

Brain inflammation is a common occurrence following responses to varied insults such as bacterial infections, stroke, traumatic brain injury and neurodegenerative disorders. A common mediator for these varied inflammatory responses is prostaglandin E2 (PGE2), produced by the enzymatic activity of cyclooxygenases (COX) 1 and 2. Previous attempts to reduce neuronal inflammation through COX inhibition, by use of nonsteroidal anti-inflammatory drugs (NSAIDs), have met with limited success. We are proposing the two-hit model for neuronal injury-an initial localized inflammation mediated by PGE2 (first hit) and the simultaneous release of adenosine triphosphate (ATP) by injured cells (second hit), which significantly enhances the inflammatory response through increased synthesis of PGE2. Several evidences on the role of exogenous ATP in inflammation have been reported, including contrary instances where extracellular ATP reduces inflammatory events. In this review, we will examine the current literature on the role of P2 receptors, to which ATP binds, in modulating inflammatory reactions during neurodegeneration. Targeting the P2 receptors, therefore, provides a therapeutic alternative to reduce inflammation in the brain. P2 receptor-based anti-inflammatory drugs (PBAIDs) will retain the activities of essential COX enzymes, yet will significantly reduce neuroinflammation by decreasing the enhanced production of PGE2 by extracellular ATP.

The anti-inflammatory effects of the 5-HT3 receptor antagonist tropisetron are mediated by the inhibition of p38 MAPK activation in primary human monocytes.

BACKGROUND: There is evidence from human and animal research that 5-hydroxytryptamine (5-HT) 3 receptor antagonists, particularly tropisetron, exert analgesic and anti-inflammatory activity. We have demonstrated that tropisetron inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)alpha and interleukin-(IL-)1beta release in primary human monocytes. The underlying mechanisms of these effects have not been investigated in detail so far. METHODS: The molecular mechanisms of the anti-inflammatory effects of tropisetron were investigated in human primary monocytes in vitro by studying IL-1beta and TNFalpha mRNA levels by PCR and reporter gene assay and by elucidating the phosphorylation of p38 mitogen activated kinase (MAPK) by Western blot. RESULTS: The steady state levels of IL-1beta and TNFalpha mRNA in LPS-activated human peripheral monocytes and the transcriptional activity of the TNFalpha promoter were not inhibited by tropisetron, suggesting that the inhibitory activity of this 5-HT3 receptor antagonist takes place at the post-transcriptional level. Additionally, we found that tropisetron prevents the phosphorylation and thus activation of the p38 MAPK, which is involved in post-transcriptional regulation of various cytokines. CONCLUSION: Our data indicate that the anti-inflammatory effects of the 5-HT3 receptor antagonist tropisetron, as shown in vivo, are possibly mediated by a selective inhibition of pro-inflammatory cytokines at the post-transcriptional level. 5-HT3 receptor antagonists are therefore a new and promising therapeutic option. New and more selective--in respect to the 5-HT3 subtypes--5-HT3R antagonists might be a future perspective in the pharmacological treatment of inflammatory diseases.

Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells.

BACKGROUND: Periventricular white matter injury (PWMI) is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs) are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs) were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2) or hypoxia (1% or 4% O(2)) for up to 7 days. We observed that 1% O(2) lead to an increase in the proportion of myelin basic protein (MBP)-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha)-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1) and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.

Protective effects of caffeine on chronic hypoxia-induced perinatal white matter injury.

OBJECTIVE: Periventricular white matter injury (PWMI) is the major cause of cerebral palsy and cognitive impairment in prematurely born infants. PWMI is characterized by reductions in cerebral myelination and cerebrocortical volumes and is associated with secondary ventriculomegaly. In neonatal rodents, these features of PWMI can be induced by rearing in chronic hypoxia or by activation of A1 adenosine receptors. We determined: (1) whether altered maturation or development of one or more oligodendrocyte (OL) lineage stages plays a role in the pathogenesis of the myelination disturbances associated with exposure to chronic hypoxia, and (2) whether blockade of A1 adenosine receptor action with the adenosine antagonist caffeine can prevent hypoxia-induced white matter injury. METHODS: Ventriculomegaly and reduced cerebral myelination were generated in mice reared in hypoxia (10% oxygen) from postnatal days 3 (P3) through 12. RESULTS: Hypomyelination was related to abnormal OL lineage progression and a reduction in the OL progenitor pool. Myelination was enhanced and ventriculomegaly reduced in hypoxia-exposed neonatal pups treated with caffeine from P3 to P12. INTERPRETATION: These observations support that hypoxia inhibits OL maturation and that caffeine administration during early postnatal development may have utility in the prevention of PWMI.

Modulation of catecholamine release from rat striatal slices by the fixed combination of aspirin, paracetamol and caffeine.

The fixed combination of aspirin, paracetamol (acetaminophen) and caffeine has been used successfully to treat different kinds of pain including migraine attacks. Even when this formulation has been marketed for a long time, the exact molecular mechanisms underlying its therapeutic effectiveness have not been completely elucidated. In the present investigation, we have studied the effects of the fixed combination of aspirin, paracetamol and caffeine (APC) on the release of dopamine and noradrenaline from rat striatal slices in an attempt to find potential new mechanisms of action of this widely used analgesic combination. We found that APC produced a significant reduction in extracellular dopamine and a dramatic increase in norepinephrine release from the slices incubated with different concentrations of APC (dose relationship 1:1:0.2, corresponding to the dose-relationship of Thomapyrin). These findings suggest that the modulation of catecholaminergic neurotransmission is a new pharmacological effect of APC which could explain the mechanism of action of this formulation, considering that the independent effect of either compound alone does not explain the potent antinociceptive properties when observed in combination.

Interleukin-1 beta-induced expression of the prostaglandin E-receptor subtype EP3 in U373 astrocytoma cells depends on protein kinase C and nuclear factor-kappaB.

Both interleukin-1beta (IL-1beta) and prostaglandins (PGs) are important mediators of physiological and pathophysiological processes in the brain. PGE2 exerts its effects by binding to four different types of PGE2 receptors named EP1-EP4. EP3 has found to be expressed in neurons, whereas expression of EP3 in glial cells has not been reported in the brain yet. Here we describe IL-1beta-induced EP3 receptor expression in human astrocytoma cells, primary astrocytes of rat and human origin and in rat brain. Using western blot, we found a marked up-regulation of EP3 receptor synthesis in human and rat primary glial cells. Intracerebroventricular administration of IL-1beta stimulated EP3 receptor synthesis in rat hippocampus. The analysis of involved signal transduction pathways by pathway-specific inhibitors revealed an essential role of protein kinase C and nuclear factor-kappaB in astrocytic IL-1beta-induced EP3 synthesis. Our data suggest that PGE2 signaling in the brain may be altered after IL-1beta release due to up-regulation of EP3 receptors. This might play an important role in acute and chronic conditions such as cerebral ischemia, traumatic brain injury, HIV-encephalitis, Alzheimer's disease and prion diseases in which a marked up-regulation of IL-1beta is followed by a prolonged increase of PGE2 levels in the brain.

Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia.

Microglia are the major cell type involved in neuroinflammatory events in brain diseases such as encephalitis, stroke, and neurodegenerative disorders, and contribute significantly to the release of prostaglandins (PGs) during neuronal insults. In this report, we studied the immediate-early intracellular signalling pathways in microglia, following bacterial lipopolysaccharide (LPS) stimulation, leading to the synthesis and release of PGE2. Here we show that LPS induces cyclooxygenase (COX) 2 by activating sphingomyelinases leading to the release of ceramides, which in turn, activate the p38 mitogen-activated protein kinases (MAPK), but not the p42/44 MAPK. We further show that exogenously added ceramide analogue (C2-ceramide) also induce PGE2 synthesis through a p38 MAPK-dependent pathway. This potential nature of ceramides in activating microglia suggests that endogenously produced ceramides during neuronal apoptosis in ischemia or neurodegenerative diseases could also contribute to the amplification of neuroinflammatory events. In contrast to protein kinase C (PKC) and phosphocholine-specific phospholipase C (PC-PLC), which transcriptionally regulate LPS-induced COX-2 synthesis, inhibition of phospholipase A2 (PLA2) has no effect on COX-2 transcription, although it inhibits the release of PGE2. Transcriptional regulation of LPS-induced COX-2 by PKC is further proved by the ability of the PKC inhibitor, Gö 6976, to inhibit LPS-induced 8-isoprostane synthesis, but not affecting LPS-induced COX-2 activity. Our data with 8-isoprostane also indicates that COX-2 plays a major role in ROS production in LPS-activated microglia. This detailed view of the intracellular signaling pathway in microglial activation and COX-2 expression opens a new therapeutic window in the search for new and more effective central anti-inflammatory agents.

Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells.

Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells.

Valproic acid inhibits substance P-induced activation of protein kinase C epsilon and expression of the substance P receptor.

The neuropeptide substance P (SP) has been hypothesized to be involved in the etiopathology of affective disorders. This hypothesis is based on the findings that neurokinin-1-receptor antagonists have antidepressant effects in depressed patients and that SP may worsen mood. In this study, we investigated the effect of the mood-stabilizing agents valproic acid (VPA), carbamazepine, and lithium on SP-induced gene expression. As a model system, we used primary rat astrocytes and human astrocytoma cells, which both express functional SP-receptors and, upon stimulation with SP, synthesize interleukin-6 (IL-6), a cytokine which has been shown to be elevated during the acute depressive state. We found that VPA dose-dependently inhibited SP-induced IL-6 synthesis which was seen with pre-incubation periods of 30 min, 3, 7 and 14 days, whereas carbamazepine and lithium showed no inhibitory effect. The inhibitory effect of VPA was not mediated by inhibition of the stress-regulated kinases p38 and p42/44 (Erk1/2) but by inhibition of protein kinase C epsilon activation. Furthermore, VPA down-regulated the expression of the substance P receptor (neurokinin(NK)-1-receptor) as assessed by real-time PCR. Whether both mechanisms contribute to the mood-stabilizing properties of VPA has to be evaluated in further studies.

1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline-induced apoptosis in the human neuroblastoma cell line SK-N-SH.

Trichloroethylene, a common industrial solvent and a metabolic precursor of chloral hydrate, occurs widely in the environment. Chloral hydrate, which is also used as a hypnotic, has been found to condense spontaneously with tryptamine, in vivo, to give rise to a highly unpolar 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) that has a structural analogy to the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Earlier studies have revealed the relative permeability of the molecule through the blood-brain barrier and its ability to induce Parkinson-like symptoms in rats. In this study, we report that TaClo induces an apoptotic pathway in the human neuroblastoma cell line, SK-N-SH, involving the translocation of mitochondrial cytochrome c to the cytosol and activation of caspase 3. TaClo-induced apoptosis shows considerable differences from that mediated by other Parkinson-inducing agents such as MPTP, rotenone and manganese. Although it is not clear if the clinically administered dosage of chloral hydrate or the relatively high environmental levels of trichloroethylene could lead to an onset of Parkinson's disease, the spontaneous in vivo formation of TaClo and its pro-apoptotic properties, as shown in this report, should be considered.