Anti-bipolar Drugs Research Articles

Mechanisms of action of anti-bipolar drugs

Mechanisms of action of anti-bipolar drugs

Regulation of Glycogen Content in Astrocytes via Cav-1/PTEN/AKT/GSK-3β Pathway by Three Anti-bipolar Drugs.

Here we present the data indicating that chronic treatment with three antibipolar drugs, lithium, carbamazepine and valproic acid regulates Cav-1/PTEN/PI3K/AKT/GSK-3β signalling pathway and glycogen content in primary cultured astrocytes. All three drugs down-regulate gene expression of Caveoline 1 (Cav-1), decrease membrane content of phosphatase and tensin homolog (PTEN), increase activity of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serine-threonine kinase (AKT), and elevate glycogen synthase kinase 3β (GSK-3β) phosphorylation thus suppressing its activity. As expected, treatment with any of these three drugs increases glycogen content in astrocytes. Our findings indicate that regulation of glycogen content via Cav-1/PTEN/AKT/GSK-3β pathway by the three anti-bipoar drugs may be responsible for therapeutic effects of these drugs, and Cav-1 is an important signal element that may contribute to pathogenesis of various CNS diseases and regulation of its gene expression may be one of the underlying mechanisms of drug action for antibipolar drugs and antidepressants currently in clinical use.

Chronic Treatment with Anti-bipolar Drugs Down-Regulates Gene Expression of TRPC1 in Neurones.

In the brain, TRPC1 channels are abundantly expressed in neurones virtually in all regions; these proteins function as receptor-activated ion channels and are implicated in numerous processes, being specifically important for neurogenesis. Primary cultures of mouse cerebellar granule cell, cerebral cortical neurones, and freshly isolated neurones from in vivo brains were used to study effects of chronic treatment with anti-bipolar drugs [carbamazepine (CBZ), lithium salts and valproic acid] on gene expression of TRPC1. Expression of TRPC1 mRNA was identified with reverse transcription-polymerase chain reaction, whereas protein content was determined by Western blotting. Store-operated plasmalemmal Ca2+ entry (SOCE) was measured with fura-2 based microfluorimetry. Chronic treatment with each of the three drugs down-regulated mRNA and protein expression in cultured cerebellar granule cells in a time- and concentration-dependent manner. Similar effect was also observed in cultured cerebral cortical neurones treated with CBZ, lithium salts and valproic acid and in freshly isolated neurones from the brains of CBZ-treated animals. The amplitude of SOCE was substantially decreased in cerebellar granule cells chronically treated with each of the three drugs. Our findings indicate that down-regulation of TRPC1 gene expression and function in neurones may be one of the mechanisms of anti-bipolar drugs action.

Evidence that changes the way you practice Bipolar Disorder: Mania and depression explained

Bipolar Disorder (BD) is characterised by alternating discrete episodes of depression and mania.1 Rational pharmacotherapy necessitates an appreciation of these different phases and of the possible underlying pathophysiology. A greater understanding of the pathogenesis of BD has boosted awareness of how anti-bipolar drugs work, and vice versa.2 This bidirectional relationship has amplified knowledge in both disciplines.

Chronic treatment with anti-bipolar drugs suppresses glutamate release from astroglial cultures.

Astroglial cells are fundamental elements of most neurological diseases, including bipolar disorders in which astrocytes show morphological and functional deficiency. Here we report the suppression of astroglial glutamate release by chronic treatment with three anti-bipolar drugs, lithium salt (Li(+)), carbamazepine (CBZ) and valproic acid (VPA). Release of glutamate was triggered by transient exposure of astrocytes to ATP (which activated purinoceptors) and 45 mM K(+) (which depolarised cell membrane to ~-30 mV). In both types of stimulation glutamate release was regulated by Ca(2+) entry through plasmalemmal channels and by Ca(2+) release from the endoplasmic reticulum (ER) intracellular stores. Exposure of astroglial cultures to Li(+), CBZ and VPA for 2 weeks led to a significant (more than 2 times) inhibition of glutamate release, which may alleviate the hyperactivity of the glutamatergic transmission in the brain of patients with bipolar disorders and thus contribute the underlying mechanism of drug action.

Fluoxetine and all other SSRIs are 5-HT2B Agonists - Importance for their Therapeutic Effects.

Fluoxetine and other serotonin-specific re-uptake inhibitors (SSRIs) are generally thought to owe their therapeutic potency to inhibition of the serotonin transporter (SERT). However, research in our laboratory showed that it affects, with relatively high affinity the 5-HT2B receptor in cultured astrocytes; this finding was confirmed by independent observations showing that fluoxetine loses its ability to elicit SSRI-like responses in behavioral assays in mice in which the 5-HT2B receptor was knocked-out genetically or inhibited pharmacologically. All clinically used SSRIs are approximately equipotent towards 5-HT2B receptors and exert their effect on cultured astrocytes at concentrations similar to those used clinically, a substantial difference from their effect on SERT. We have demonstrated up-regulation and editing of astrocytic genes for ADAR2, the kainate receptor GluK2, cPLA2 and the 5-HT2B receptor itself after chronic treatment of cultures, which do not express SERT and after treatment of mice (expressing SERT) for 2 weeks with fluoxetine, followed by isolation of astrocytic and neuronal cell fractionation. Affected genes were identical in both experimental paradigms. Fluoxetine treatment also altered Ca2+ homeostatic cascades, in a specific way that differs from that seen after treatment with the anti-bipolar drugs carbamazepine, lithium, or valproic acid. All changes occurred after a lag period similar to what is seen for fluoxetine’s clinical effects, and some of the genes were altered in the opposite direction by mild chronic inescapable stress, known to cause anhedonia, a component of major depression. In the anhedonic mice these changes were reversed by treatment with SSRIs.

Signal Transduction in Astrocytes during Chronic or Acute Treatment with Drugs (SSRIs, Antibipolar Drugs, GABA-ergic Drugs, and Benzodiazepines) Ameliorating Mood Disorders

Chronic treatment with fluoxetine or other so-called serotonin-specific reuptake inhibitor antidepressants (SSRIs) or with a lithium salt “lithium”, carbamazepine, or valproic acid, the three classical antibipolar drugs, exerts a multitude of effects on astrocytes, which in turn modulate astrocyte-neuronal interactions and brain function. In the case of the SSRIs, they are to a large extent due to 5-HT2B-mediated upregulation and editing of genes. These alterations induce alteration in effects of cPLA2, GluK2, and the 5-HT2B receptor, probably including increases in both glucose metabolism and glycogen turnover, which in combination have therapeutic effect on major depression. The ability of increased levels of extracellular K+ to increase [Ca2+]i is increased as a sign of increased K+-induced excitability in astrocytes. Acute anxiolytic drug treatment with benzodiazepines or GABAA receptor stimulation has similar glycogenolysis-enhancing effects. The antibipolar drugs induce intracellular alkalinization in astrocytes with lithium acting on one acid extruder and carbamazepine and valproic acid on a different acid extruder. They inhibit K+-induced and transmitter-induced increase of astrocytic [Ca2+]i and thereby probably excitability. In several cases, they exert different changes in gene expression than SSRIs, determined both in cultured astrocytes and in freshly isolated astrocytes from drug-treated animals.

Chronic fluoxetine administration increases expression of the L-channel gene Cav1.2 in astrocytes from the brain of treated mice and in culture and augments K+-induced increase in [Ca2+

We have recently shown that freshly isolated astrocytes from the mouse brain express mRNA for the L-channel gene Cav1.3 to at least the same degree (per mg mRNA) as corresponding neurons. The amount of extracellular Ca2+ actually entering cultured astrocytes by its opening is modest, but due to secondary Ca2+-mediated stimulation of the ryanodine receptor (RyR) the increase in free cytosolic Ca2+ [Ca2+]i is substantial. The other Cav1 subtype expressed in brain is Cav1.2, which is even expressed in higher density. Although the different primers used for the two genes preclude exact quantitative comparison, the present study suggests that this is also the case in the freshly isolated astrocytes and neurons, which express equal Cav1.2 densities. Again, most of the increase in [Ca2+]i occurred by RyR activity. In contrast to Cav1.3 the expression of Cav1.2 was greatly increased (doubled) after two weeks of treatment with fluoxetine hydrochloride (10mg/kg). Accordingly [Ca2+]i in cultured astrocytes exposed to the addition of 10–60mM KCl increased substantially in cultured astrocytes treated chronically with fluoxetine with the lag time until the effect was observed depending upon the fluoxetine concentration. This effect was inhibited by nifedipine or siRNA against Cav1.2. The increase in K+-induced rise in [Ca2+]i after fluoxetine treatment is directly opposite to a decrease in [Ca2+]i after treatment with any of the anti-bipolar drugs lithium, carbamazepine or valproic acid, due to reduced capacitative Ca2+ influx. We have previously shown a similar effect after fluoxetine treatment, but it becomes overridden by the Cav1.2 up-regulation.

Comparison between drug-induced and K+-induced changes in molar acid extrusion fluxes (JH +) and in energy consumption rates in astrocytes

Neuronal excitation leads to an increase of the extracellular K(+) concentration ([K(+)]o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pHi) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na(+)/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na(+), K(+)-ATPase); and (2) activation of cellular reuptake of K(+) mediated by the Na(+), K(+)-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pHi is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third 'classical' anti-bipolar drug, 'lithium' increases astrocytic pHi by a different mechanism (stimulation of the acid extruding Na(+)/H(+) exchanger NHE1). The acid extruder fluxes, which depend upon the change in pHi per time unit (ΔpHi/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pHi was determined using the fluorescent pH-sensitive indicator BCECF-AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pHi changes/min during recovery after acid-loading with NH3/NH4 (+), NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K(+) concentration. Energetically, K(+) uptake is much costlier than NBCe1 activity.

Mechanisms for L-channel-mediated increase in [Ca2+]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels

The importance of Ca2+ signaling in astrocytes is undisputed but a potential role of Ca2+ influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca2+ concentration ([Ca2+]i) in astrocytes in primary cultures in response to an increased extracellular K+ concentration (45mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca2+, known to occur in response to an elevation in [Ca2+]i. This means that the actual influx of Ca2+ is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca2+ currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K+-mediated increase in [Ca2+]i. This is shown to be due to an inhibition of capacitative Ca2+ influx, reflected by decreased mRNA and protein expression of the ‘transient receptor potential channel’ (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca2+ influx in response to an elevated extracellular K+ concentration is important for generation of Ca2+ oscillations and for the stimulatory effect of elevated K+ concentrations in intact, non-cultured brain tissue, and (ii) that Ca2+ channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Cav1.3 is demonstrated in freshly separated astrocytes from normal brain.

Cell-Specific mRNA Alterations in Na+, K+-ATPase α and β Isoforms and FXYD in Mice Treated Chronically with Carbamazepine, an Anti-Bipolar Drug

Evidence accumulating during almost 50 years suggests Na(+), K(+)-ATPase dysfunction in bipolar disorder, a disease treatable with chronic administration of lithium salts, carbamazepine or valproic acid. Three Na(+), K(+)-ATPase α subunits (α1-3) and two β subunits (β1 and β2) are expressed in brain together with the auxiliary protein FXYD7. FXYD7 decreases K(+) affinity, and thus contributes to stimulation of the enzyme at elevated extracellular K(+) concentrations. Na(+), K(+)-ATPase subtype and FXYD7 genes were determined by RT-PCR in mice co-expressing one fluorescent signal with an astrocytic marker or a different fluorescent signal with a neuronal marker and treated for 14 days with carbamazepine. Following fluorescence-activated cell sorting of neurons and astrocytes it was shown that α2 Expression was upregulated in astrocytes and neurons and α1 selectively in neurons, but α3 was unchanged. β1 was upregulated in astrocytes, but not in neurons. β2 was unaffected in astrocytes and absent in neurons. FXYD7 was downregulated specifically in neurons. According to cited literature data these changes should facilitate K(+) uptake in neurons, without compromising preferential uptake in astrocytes at increased extracellular K(+) concentrations. This process seems to be important for K(+) homeostasis of the cellular level of the brain (Xu et al. Neurochem Res E-pub Dec. 12, 2012).

Chronic Treatment with Anti-bipolar Drugs Causes Intracellular Alkalinization in Astrocytes, Altering Their Functions

Bipolar disorder I and II are affective disorders with mood changes between depressive and manic (bipolar I) or hypomanic (bipolar II) periods. Current therapy of these conditions is chronic treatment with one or more of the anti-bipolar drugs, Li(+) ('lithium'), carbamazepine and valproic acid. The pathophysiology of bipolar disorder is multifactorial and far from clear. Recent data on the dependence of normal brain function on neuronal-astrocytic interactions raise the possibility of astrocytic involvement. We will discuss our previously published and new results on effects of chronic treatment of primary cultures of normal mouse astrocytes with any of three conventional anti-bipolar drugs. The focus will be on several drug-induced events in relation to therapeutic effects of the drugs, such as myo-inositol uptake, intracellular pH and alkalinization, drug-induced modulation of glutamatergic activity in astrocytes and release of astrocytic 'gliotransmitters'. Finally, we will discuss the importance of phospholipase A2 (PLA(2)) and arachidonic acid cascade in drug-treated astrocytes, partly based on Dr. Barneda Cuirana's published thesis. All three drugs cause gradual intracellular alkalinization through different mechanisms. Alkalinization inhibit myo-inositol uptake, resulting in reduced inositolphosphate/phospholipid signaling. Accordingly, transmitter-induced increase in free intracellular Ca(2+) ([Ca(2+)](i)) becomes inhibited, aborting release of astrocytic 'gliotransmitters'. The reduction of "gliotransmitter" effects on neurons may have therapeutic effects in mania. Alkalinization also up-regulates expression of cPLA(2), an enzyme releasing arachidonic acid, and triggered arachidonic acid cascade and production, but perhaps not release, of prostaglandins. Whenever tested, identical effects were observed in freshly isolated astrocytes, but not neurons, from carbamazepine-treated healthy animals.

Yeast bioassay for identification of inositol depleting compounds

Bipolar affective disorder is a chronic, severe, debilitating illness affecting 1–2% of the population. Valproate, along with lithium and carbamazepine, are the only drugs for which long-term efficacy has been established. However, these drugs are ineffective for, and not well tolerated by, a large number of patients and are also associated with teratogenicity and reproductive defects. Therefore, there is a substantial need to develop more effective anti-bipolar drugs. We have previously shown that valproate, like lithium, decreases intracellular inositol, which supports the inositol depletion hypothesis. We employed inositol depletion in yeast as a screening tool to identify potential new anti-bipolar medications. We show here that hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, ethylhexanoate, and methyloctanoate decrease intracellular inositol levels and increase the expression of INO1, the gene encoding myo-inositol-3-phosphate synthase (MIPS). Similar to valproate, these inositol-depleting carboxylic acids inhibited MIPS indirectly. A correlation was shown between cell growth inhibition and the increase in INO1 expression by the carboxylic acids, factors that were reversed in the presence of inositol. Inositol depletion in yeast may be exploited as an easy and inexpensive screening test for potential new inositol depleting anti-bipolar drugs.

Chronic N-methyl-d-aspartate administration increases the turnover of arachidonic acid within brain phospholipids of the unanesthetized rat

Whereas antibipolar drug administration to rats reduces brain arachidonic acid turnover, excessive N-methyl-d-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms and may increase arachidonic acid turnover in rat brain phospholipids. To determine whether chronic NMDA would increase brain arachidonic acid turnover, rats were daily administered NMDA (25 mg/kg, ip) or vehicle for 21 days. In unanesthetized rats, on day 21, [1-(14)C]arachidonic acid was infused intravenously and arterial blood plasma was sampled until the animal was euthanized at 5 min and its microwaved brain was subjected to chemical and radiotracer analysis. Using equations from our in vivo fatty acid model, we found that compared with controls, chronic NMDA increased the net rate of incorporation of plasma unesterified arachidonic acid into brain phospholipids (25-34%) as well as the turnover of arachidonic acid within brain phospholipids (35-58%). These changes were absent at 3 h after a single NMDA injection. The changes, opposite to those after chronic administration of antimanic drugs to rats, suggest that excessive NMDA signaling via arachidonic acid may be a model of upregulated arachidonic acid turnover in brain phospholipids.

Up-regulation of cPLA2 gene expression in astrocytes by all three conventional anti-bipolar drugs is drug-specific and enzyme-specific

Common biological effects by all three conventional anti-bipolar drugs, the lithium ion (Li(+)), carbamazepine, and valproic acid, are important because identical effects may provide information about the pathophysiology of affective disorders. It has been reported that chronic treatment with either drug in vivo down-regulates the turnover of arachidonic acid in brain. This reaction is catalyzed by Ca(2+)-dependent phospholipase A(2) (cPLA(2)), the expression of which was down-regulated by Li(+) or carbamazepine but not by valproic acid; expression of two other PLA subtypes, iPLA(2) and sPLA(2) was unaffected. cPLA(2) is amply expressed in astrocytes, and in the present study, effects of 1-4 weeks of treatment with clinically relevant concentrations of each of the three anti-bipolar drugs on cPLA(2), iPLA(2), and sPLA(2) mRNA and protein expression were determined in primary cultures of mouse astrocytes by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. Two or more weeks treatment with Li(+) concentrations below 2 mM, carbamazepine or valproic acid up-regulated mRNA and protein expression of cPLA(2), but had no effect on iPLA(2) and sPLA(2), showing enzyme specificity. The effect occurred more rapidly at higher than lower concentrations but also tended to end after 4 weeks at the higher concentrations. Two millimolar Li(+) caused an initial increase of cPLA(2) followed by a decrease after 3 and 4 weeks. Topiramate had no effect, indicating specificity for anti-bipolar drugs. Both up- and down-regulation of cPLA(2) gene expression are involved in the mechanisms of action of anti-bipolar drugs; astrocytes are a target for these drugs.

Genetic Perturbation of Glycolysis Results in Inhibition of de Novo Inositol Biosynthesis

In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the "inositol-less death" phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis.

Chronic Carbamazepine Decreases the Incorporation Rate and Turnover of Arachidonic Acid but Not Docosahexaenoic Acid in Brain Phospholipids of the Unanesthetized Rat: Relevance to Bipolar Disorder

The basis for carbamazepine's efficacy in treating bipolar disorder is not agreed on. One hypothesis is that, similar to lithium and valproate (antibipolar drugs), carbamazepine might selectively decrease the kinetics of arachidonic acid (AA) in brain phospholipids. To assess whether it targets brain AA kinetics, we administered carbamazepine (25 mg/kg/day, IP) to rats for 30 days and then determined its effect compared with that of vehicle on incorporation and turnover rates of AA and docosahexaenoic acid (DHA) in brain phospholipids. In unanesthetized rats that had received carbamazepine or vehicle, [1-14C]AA or [1-14C]DHA was infused intravenously, and arterial blood plasma was sampled until the animal was killed at 5 min and its brain, after being microwaved, was used for acyl-coenzyme A (acyl-CoA) and phospholipid fatty acid analysis. Chronic carbamazepine, compared with vehicle, decreased the rate of incorporation of AA-CoA (27%-29%) and turnover of AA (25%-27%) but not of DHA-CoA or DHA in brain phospholipids. The results, which are comparable to published findings after chronic administration of lithium and valproic acid to rats, support the hypothesis that drugs effective against mania in bipolar disorder act by selectively downregulating the incorporation rate of AA-CoA and turnover of AA in brain phospholipids.

Alteration of Lithium Pharmacology through Manipulation of Phosphoadenosine Phosphate Metabolism

Bisphosphate 3'-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium. Here we report a role for 3'-nucleotidase and its substrate, 3'-phosphoadenosine 5'-phosphate (PAP), in mediating the cellular effects of lithium. Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1. Disruption of the yeast 3'-nucleotidase gene or treatment of cells with lithium results in a >80-fold accumulation of PAP and leads to potent growth inhibition. These data indicate that the accumulation of a 3'-nucleotidase substrate, such as PAP, mediates the toxicity of lithium. To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly down-regulated. Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional down-regulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of these effects were reversed by heterologous expression of human sulfurylase and kinase. Collectively, our data support a role for 3'-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for the treatment of bipolar disorder.

Human 1-D-myo-Inositol-3-phosphate Synthase Is Functional in Yeast

We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1). The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 degrees C and exhibited K(m) values of 0.57 mm and 8 microm for glucose 6-phosphate and NAD(+), respectively. NH(4)(+) and K(+) were better activators than other cations tested (Na(+), Li(+), Mg(2+), Mn(2+)), and Zn(2+) strongly inhibited activity. Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase (ino1Delta/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.

Regulation of c-Jun N-terminal kinase, p38 kinase and AP-1 DNA binding in cultured brain neurons: roles in glutamate excitotoxicity and lithium neuroprotection.

In rat cerebellar granule cells, glutamate induced rapid activation of c-Jun N-terminal kinase (JNK) and p38 kinase to phosphorylate c-Jun (at Ser63) and p53 (at Ser15), respectively, and a subsequent marked increase in activator protein-1 (AP-1) binding that preceded apoptotic death. These glutamate-induced effects and apoptosis could largely be prevented by long-term (7 days) pretreatment with 0.5-2 mm lithium, an antibipolar drug. Glutamate's actions could also be prevented by known blockers of this pathway, MK-801 (an NMDA receptor blocker), SB 203580 (a p38 kinase inhibitor) and curcumin (an AP-1 binding inhibitor). The concentration- and time-dependent suppression of glutamate's effects by lithium and curcumin correlated well with their neuroprotective effects. These results suggest a prominent role of JNK and p38, as well as their downstream AP-1 binding activation and p53 phosphorylation in mediating glutamate excitotoxicity. Moreover, the neuroprotective effects of lithium are mediated, at least in part, by suppressing NMDA receptor-mediated activation of the mitogen-activated protein kinase pathway.