Effect of PPM1F in dorsal raphe 5-HT neurons in regulating methamphetamine-induced conditioned place preference performance in mice.

Methamphetamine (METH), a synthetically produced central nervous system stimulant, is one of the most illicit and addictive drugs worldwide. Protein phosphatase Mg2 + /Mn2 + -dependent 1F F (PPM1F) has been reported to exert multiple biological and cellular functions. Nevertheless, the effects of PPM1F and its neuronal substrates on METH addiction remain unclear. Herein, we first established a METH-induced conditioned place preference (CPP) mouse model. We showed that PPM1F is widely distributed in 5-HT neurons of the dorsal raphe nucleus (DRN), and METH treatment decreased the expression of PPM1F in DRN, which was negatively correlated with METH-induced CPP behaviors. Knockout of PPM1F mediated by adeno-associated virus (AAV) in DRN produced enhanced susceptibility to METH-induced CPP, whereas the overexpression of PPM1F in DRN attenuated METH-induced CPP phenotypes. The expression levels of Tryptophan hydroxylase2 (TPH2) and serotonin transporter (SERT) were down-regulated with a concurrent reduction in 5-hydroxytryptamine (5-HT), tryptophan hydroxylase2 (TPH2)-immunoreactivity neurons and 5-HT levels in DRN of PPM1F knockout mice. In the end, decreased expression levels of PPM1F were found in the blood of METH abusers and METH-taking mice. These results suggest that PPM1F in DRN 5-HT neurons regulates METH-induced CPP behaviors by modulating the key components of the 5-HT neurotransmitter system, which might be an important pathological gene and diagnostic marker for METH-induced addiction.

Curcumin Modulates the NMDA Receptor Subunit Composition Through a Mechanism Involving CaMKII and Ser/Thr Protein Phosphatases.

Curcumin is one of the major compounds contained in turmeric, the powdered rhizome of Curcuma longa. Results obtained in various experimental models indicate that curcumin has the potential to treat a large variety of neuronal diseases. Excitotoxicity, the toxicity due to pathological glutamate receptors stimulation, has been considered to be involved in several ocular pathologies including ischemia, glaucoma, and diabetic retinopathy. The NMDA receptor (NMDAR), a heteromeric ligand-gated ion channel, is composed of GluN1 and GluN2 subunits. There are four GluN2 subunits (GluN2A-D), which are major determinants of the functional properties of NMDARs. It is widely accepted that GluN2B has a pivotal role in excitotoxicity while the role of GluN2A remains controversial. We previously demonstrated that curcumin is neuroprotective against NMDA-induced excitotoxicity with a mechanism involving an increase of GluN2A subunit activity. In this paper, we investigate the mechanisms involved in curcumin-induced GluN2A increase in retinal cultures. Our results show that curcumin treatment activated CaMKII with a time-course that paralleled those of GluN2A increase. Moreover, KN-93, a CaMKII inhibitor, was able to block the effect of curcumin on GluN2A expression. Finally, in our experimental model, curcumin reduced ser/thr phosphatases activity. Using okadaic acid, a specific PP1 and PP2A blocker, we observed an increase in GluN2A levels in cultures. The ability of okadaic acid to mimic the effect of curcumin on GluN2A expression suggests that curcumin might regulate GluN2A expression through a phosphatase-dependent mechanism. In conclusion, our findings indicate curcumin modulation of CaMKII and/or ser/thr phosphatases activities as a mechanism involved in GluN2A expression and neuroprotection against excitotoxicity.

Inhibitory effect of sanguinarine on PKC-CPI-17 pathway mediating by muscarinic receptors in dispersed intestinal smooth muscle cells.

This study investigated the inhibitory effects of sanguinarine (SA) on PKC-CPI-17 pathway in rat intestinal smooth muscle cells (ISMC). Previous studies indicate that the inhibitory effects of SA on ISMC contraction are possibly mediated by the Ca(2+) influx. ISMC was treated with 1 µM SA for 24h remarkably inhibited the mRNA expression of m2 and m3 receptors. ISMC treated with 1 or 3 µM SA for 30 min significantly decreased the mRNA expression of PKC-δ, PKC-ε, PKC-η, and CPI-17. 1 µM SA could markedly inhibit carbachol (CCh)-mediated increase PKC-δ, PKC-η, and CPI-17 mRNA but had no effect in PKC-ε.Treatment of ISMC with SA (1 µM, 30 min) caused a decrease in protein expression of PKC-δ. However, the expression of CPI-17 was significantly inhibited in a time-dependent manner. These results demonstrate that the inhibitory effect of SA is coupled with alteration of PKC-mediated signal transduction and intracellular Ca(2+) concentration.

Drug design for protein kinases and phosphatases: flexible-receptor docking, binding affinity and specificity, and drug-binding kinetics.

This article reviews some of our experiences on applying computational techniques to aid the design of drugs targeting protein kinases and phosphatases. It is not a comprehensive review. Rather, it focuses on several less explored approaches or ideas that we have experiences on. It reviews some recent improvements on the Poisson-Boltzmann/Surface Area model for calculating binding affinity and discusses ways to perform calculations that are more tolerant to statistical and systematic errors. Several new ways to incorporate protein flexibility in molecular docking and estimating binding affinity are also discussed. Its discussions also go beyond binding affinity to considering drug-binding kinetics, not only on investigating protein-ligand interactions in isolation, but also on accounting for upstream and downstream influences that can occur in cells, through kinetic modeling of cell signaling. This review also describes a quick molecular simulation method for understanding drug-binding kinetics at the molecular level, with the hope of generating guiding principles for designing drugs with the desired kinetic properties. Sources of drug-binding selectivity that appear obvious but often overlooked are also discussed.

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana.

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca(2+)](cyt)) in guard cells. While abscisic acid-induced [Ca(2+)](cyt) oscillation has been extensively studied, MeJA-induced [Ca(2+)](cyt) oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca(2+)](cyt) oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca(2+) reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca(2+)](cyt) oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca(2+)](cyt) oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.

[Oxidative damage to the endoplasmic reticulum stress pathway of apoptosis-related molecules expression in MIN6 cell].

AIM: Through a third-butyl hydrogen peroxide (t-BHP) induced apoptosis in pancreatic islet ß-cells to study the oxidative damage induced endoplasmic reticulum stress-JNK pathway of apoptosis related molecules in vitro. METHODS: Mouse insulinoma(MIN6) cells was administered with t-BHP which were cultured in vitro. Choosing medicine with different concentrations(0-400 µmol/L)and time periods(0-8 h)to establish the cells apoptosis model. The percentage of cell viability was determined through CCK-8 assay. The percentage of apoptosis was determined through flow cytometric assay after Annexin-V-FITC-PI staining. The activity of caspase-3 was measured by the caspase-3 activity assay kit. The expression of Endoplasmic reticulum stress-related molecules and the apoptosis signal pathway IRE1, JNK, P-JNK, Caspase-3 were detected by Western blot. RESULTS: The percentage of MIN6 cell viability was reducing with the concentration of t-BHP increasing. The Caspase-3 significantly change the activity after exposured of t-BHP in a concentration ≥ 25 µmol/L when the role of ≥1 h, With t-BHP concentration was increased, the role of prolonged, endoplasmic reticulum stress transmembrane protein IRE1α, P-JNK, active caspase-3 expression was significantly increased. CONCLUSION: The study demonstrates that the percentage of MIN6 cell viability was reduced in a dose-dependent manner. Continuous exposuring of t-BHP induced oxidative damage in MIN6 cells to endoplasmic reticulum stress and apoptosis. The expression of Endoplasmic reticulum stress and apoptosis pathway-related molecules in cell apoptosis in a dose and time-dependent.

Luteolin inhibits cytokine expression in endotoxin/cytokine-stimulated microglia.

Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative disease by producing excessive proinflammatory cytokines and nitric oxide (NO). Luteolin, a naturally occurring polyphenolic flavonoid antioxidant, has potent anti-inflammatory and neuroprotective properties both in vitro and in vivo. However, the molecular mechanism of luteolin-mediated immune modulation in microglia is not fully understood. In the present study, we report the inhibitory effect of luteolin on lipopolysaccharide (LPS)/interferon γ (IFN-γ)-induced NO and proinflammatory cytokine production in rat primary microglia and BV-2 microglial cells. Luteolin concentration-dependently abolished LPS/IFN-γ-induced NO, tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) production as well as inducible nitric oxide synthase (iNOS) protein and mRNA expression. Luteolin exerted an inhibitory effect on transcription factor activity including nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF-1) in LPS/IFN-γ-activated BV-2 microglial cells. Biochemical and pharmacological studies revealed that the anti-inflammatory effect of luteolin was accompanied by down-regulation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), Akt and Src. Further studies have demonstrated that the inhibitory effect of luteolin on intracellular signaling execution and proinflammatory cytokine expression is associated with resolution of oxidative stress and promotion of protein phosphatase activity. Together, these results suggest that luteolin suppresses NF-κB, STAT1 and IRF-1 signaling, thus attenuating inflammatory response of brain microglial cells.

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5.

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.

Neuroprotection of gamma-aminobutyric acid receptor agonists via enhancing neuronal nitric oxide synthase (Ser847) phosphorylation through increased neuronal nitric oxide synthase and PSD95 interaction and inhibited protein phosphatase activity in cerebral ischemia.

It is well documented that exitotoxicity induced by N-methyl-D-aspartate (NMDA) receptor activation plays a pivotal role in delayed neuronal death in the hippocampal CA1 region after transient global ischemia. However, the effect of gamma-aminobutyric acid (GABA) receptor activation is uncertain in ischemia brain injury. The aim of this study was to investigate whether the enhancement of GABA receptor activity could inhibit NMDA receptor-mediated nitric oxide (NO) production by neuronal NO synthase (nNOS) in brain ischemic injury. The results showed that both the GABA(A) receptor agonist muscimol and the GABA(B) receptor agonist baclofen had neuroprotective effect, and the combination of two agonists could significantly protect neurons against death induced by ischemia/reperfusion. Coapplication of muscimol with baclofen not only enhanced nNOS (Ser847) phosphorylation but also increased the interaction of nNOS with PSD95 at 6 hr and 1 day of reperfusion. Interestingly, the inhibitors of calcineurin and PP1/PP2A could enhance nNOS phosphorylation at Ser847 site at 1 day of reperfusion after ischemia but not at 6 hr of reperfusion. From these data, we conclude that GABA receptor activation could exert its neuroprotective effect through increasing nNOS (Ser847) phosphorylation by different mechanisms at 6 hr and 1 day of reperfusion. The increased interaction of nNOS and postsynaptic density-95 induced by GABA agonists is responsible for nNOS (Ser847) phosphorylation at both time points, but at 1 day of reperfusion the inhibition of protein phosphatase activity by GABA agonists also contributes to the neuroprotection. Our results suggest that GABA receptor agonists may serve as a potential and important neuroprotectant in therapy for ischemic stroke.

Phosphoprotein analysis for investigation of in vivo relationship between protein phosphatase inhibitory activities and acute hepatotoxicity of microcystin-LR.

Microcystin-LR (MCLR) produced by freshwater cyanobacteria is a potent hepatotoxin and inhibits protein serine/threonine phosphatases 1 and 2A (PP1 and PP2A). Okadaic acid (OA) is a similar phosphatase inhibitor, which has less affinity to PP1 than PP2A. MCLR and OA behave similarly with primary culture hepatocytes with the induction of phosphorylation of the cytokeratins, morphological changes, and apoptosis. The purpose of this study was to investigate the in vivo relationship between the protein phosphatase inhibitory activities and the acute hepatotoxicity of MCLR compared to OA. MCLR and OA were intraperitoneally administrated to mice at approximately 220 microg/kg. After 30 min, the liver of only the MCLR-treated mouse was dark-colored and heavier than that of the control mouse. Subsequently, the phosphoproteins of the mouse liver were chemically modified with reversible biotinylation reagent and selectively analyzed by LC/MS/MS. Consequently, the phosphorylated Ser 354 of formyltetrahydrofolate dehydrogenase, which is an abundant enzyme in the liver cytoplasm, was observed in the MCLR- and the OA-treated mice 9.5 and 5.3 times more intensely than in the control mouse respectively, suggesting that MCLR and OA inhibited PP2A and induced the resulting phosphorylation. These results supported the hypothesis that the acute hepatotoxicity is possibly caused by the PP1 inhibition, and not by the PP2A inhibition.

Blockade of adenosine A(2A) receptors prevents staurosporine-induced apoptosis of rat hippocampal neurons.

Since adenosine A(2A) receptor (A(2A)Rs) blockade protects against noxious brain insults involving apoptosis, we directly tested if A(2A)R blockade prevents apoptosis induced by staurosporine (STS). Exposure of rat hippocampal neurons to STS (30 nM, 24 h) decreased neuronal viability while increasing the number apoptotic-like neurons and de-localizing mitochondria and cytochrome c immunoreactivities. This was prevented by the selective A(2A)R antagonists, SCH58261 and ZM241385 (50 nM). Shorter incubation periods (6 h) with STS caused no neuronal loss but decreased synaptophysin and MAP-2 immunoreactivities, which was prevented by SCH58261. Furthermore, STS (100 nM) decreased MTT reduction and increased caspase-3 activity in rat hippocampal nerve terminals, which was prevented by SCH58261. These results show that A(2A)R blockade inhibits STS-induced apoptotic-like neuronal cell death. This begins with an apoptotic-like synaptotoxicity, which later evolved into an overt neurotoxicity, and A(2A)Rs effectively control this initial synaptotoxicity, in agreement with their predominant synaptic localization in the hippocampus.

Perinatal asphyxia induces neurogenesis in hippocampus: an organotypic culture study.

There is clinical and experimental evidence indicating that neurocircuitries of the hippocampus are vulnerable to hypoxia/ischemia occurring at birth, inducing, upon re-oxygenation/re-circulation, delayed neuronal death, but also compensatory mechanisms, including neurogenesis. In the present report, perinatal asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath at 37 degrees C for 20 min. Some pups were delivered immediately after the hysterectomy to be used as non-asphyxiated caesarean-delivered controls. The pups were sacrificed after seven days for preparing organotypic hippocampal cultures. The cultures were grown on a coverslip in a medium-containing culture tube inserted in a hole of a roller device standing on the internal area of a cell incubator at 35 degrees C, 10% CO2. At days in vitro (DIV) 25-27, cultures were fixed for assaying cell proliferation and neuronal phenotype with antibodies against 5-bromo-2'deoxyuridine (BrdU) and microtubule associated protein-2 (MAP-2), respectively. Confocal microscopy revealed that there was a 2-fold increase of BrdU-positive, but a 40% decrease of MAP-2-positive cells/mm3 in cultures from asphyxia-exposed, compared to that from control animals. Approximately 30% of BrdU-positive cells were also positive for MAP-2 (approximately 4800 cells), mainly seen in the dentate gyrus of the hippocampus, demonstrating a 3-fold increase of postnatal neurogenesis, when the total amount of double-labelled cells seen in cultures from asphyxia-exposed animals is compared to that from control animals.

The presence of the cyanobacterial toxin microcystin in black band disease of corals.

Black band disease (BBD) is a migrating, cyanobacterial dominated, sulfide-rich microbial mat that moves across coral colonies lysing coral tissue. While it is known that BBD sulfate-reducing bacteria contribute to BBD pathogenicity by production of sulfide, additional mechanisms of toxicity may be involved. Using HPLC/MS, the cyanotoxin microcystin was detected in 22 field samples of BBD collected from five coral species on nine reefs of the wider Caribbean (Florida Keys and Bahamas). Two cyanobacterial cultures isolated from BBD, Geitlerinema and Leptolyngbya sp. contained microcystin based on HPLC/MS, with toxic activity confirmed using the protein phosphatase inhibition assay. The gene mcyA from the microcystin synthesis complex was detected in two field samples and from both BBD cyanobacterial cultures. Microcystin was not detected in six BBD samples from a different area of the Caribbean (St Croix, USVI) and the Philippines, suggesting regional specificity for BBD microcystin. This is the first report of the presence of microcystin in a coral disease.

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia.

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.

Impaired long-term depression in P2X3 deficient mice is not associated with a spatial learning deficit.

The hippocampus is a brain region critical for learning and memory processes believed to result from long-lasting changes in the function and structure of synapses. Recent findings suggest that ATP functions as a neurotransmitter or neuromodulator in the mammalian brain, where it activates several different types of ionotropic and G protein-coupled ATP receptors that transduce calcium signals. However, the roles of specific ATP receptors in synaptic plasticity have not been established. Here we show that mice lacking the P2X3 ATP receptor (P2X3KO mice) exhibit abnormalities in hippocampal synaptic plasticity that can be restored by pharmacological modification of calcium-sensitive kinase and phosphatase activities. Calcium imaging studies revealed an attenuated calcium response to ATP in hippocampal neurons from P2X3KO mice. Basal synaptic transmission, paired-pulse facilitation and long-term potentiation are normal at synapses in hippocampal slices from P2X3KO. However, long-term depression is severely impaired at CA1, CA3 and dentate gyrus synapses. Long-term depression can be partially rescued in slices treated with a protein phosphatase 1-2 A activator or by postsynaptic inhibition of calcium/calmodulin-dependent protein kinase II. Despite the deficit in hippocampal long-term depression, P2X3KO mice performed normally in water maze tests of spatial learning, suggesting that long-term depression is not critical for this type of hippocampus-dependent learning and memory.

Lithium-induced activation of Akt and CaM kinase II contributes to its neuroprotective action in a rat microsphere embolism model.

Lithium used in bipolar mood disorder therapy protects neurons from brain ischemic cell death. Here, we documented that lithium administration under microsphere-embolism (ME)-induced brain ischemia restored decreased protein kinase B (Akt) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activities 24 h after ischemia in rat brain. Akt activation was associated with increased phosphorylation of its potential targets forkhead transcription factor (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). In parallel with decreased CaMKII autophosphorylation, we also found marked dephosphorylation of tau proteins 24-72 h after ME. Increased protein phosphatase 2A (PP2A) activity was found 24 h after ME. Inhibition of increased PP2A activity by lithium treatment apparently mediated restored tau phosphorylation. Taken together, activation of Akt and CaMKII by lithium was associated with neuroprotective activity in ME-induced neuronal injury.

A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1.

G(q) protein-coupled receptor stimulation increases sarcolemmal Na(+)/H(+) exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A(1) receptor stimulation inhibits this response through a G(i) protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A(1) receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the alpha(1)-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A(c)) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G(i) proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A(c) and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A(c) dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A(1) receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G(i) protein-mediated translocation of PP2A(c) and NHE1 dephosphorylation.

Effects of neonatal dexamethasone treatment on hippocampal synaptic function.

OBJECTIVE: Synthetic glucocorticoid dexamethasone (DEX) is frequently used as a therapeutic agent to lessen the morbidity of chronic lung disease in premature infants. Surprisingly, little is known about the long-term neurodevelopmental outcomes of this therapy. METHODS: Using a schedule of tapering doses of DEX similar to that used in premature infants, we examined the consequences of neonatal DEX treatment on hippocampal synaptic plasticity of infants and associative memory later in their lives. RESULTS: Neonatal DEX treatment changed the direction of synaptic plasticity, favoring low-frequency, stimulation-induced, long-term depression and opposing the induction of long-term potentiation by high-frequency stimulation in adolescent (5-week-old) rats, but these alterations disappeared in young adult (8-week-old) rats. The effects of DEX on long-term depression and long-term potentiation were found to correlate with an increase in the autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase II and a decrease in the protein phosphatase 1 activity. Neonatal DEX treatment also disrupted memory retention in 5-week-old (but not 8-week-old) rats subjected to passive avoidance learning tasks. INTERPRETATION: These results suggest that neonatal DEX treatment alters hippocampal synaptic plasticity and contextual fear memory formation in later life, but these impairments apparently are not permanent.

Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by arsenic sulphide: involvement of serine/threonine protein phosphatases.

The clinical efficacy of arsenic sulfide (As(4)S(4)), also known as realgar, in the treatment of leukemia in China is prompting people to explore the underlying mechanism. We examined the realgar-induced differentiation in human promyelocytic leukemia cell line HL-60. Cells exhibited proliferation inhibition when treated with 0.10-1.5 microM of realgar, and underwent monocytic differentiation as indicated by morphological changes, NBT reduction assay, and cytofluorometric analyses of the cell surface antigens, CD11b and CD14. Accompanying the differentiation, the activity of serine/threonine protein phosphatase type 1 (PP1) and type 2A (PP2A) were enhanced, whereas the activity of PP2B remained virtually the same compared to the control. When cells were treated with realgar in the presence of an inhibitor of PP1 and 2A or an inhibitor of PP2B, the differentiation of the cells was partially suppressed as revealed by NBT reduction assay and the expression of CD14. Our data demonstrate that realgar induces monocytic differentiation in HL-60 cells and that some serine/threonine protein phosphatases may be involved in the process.

Pathological effects of glyoxalase I inhibition in SH-SY5Y neuroblastoma cells.

In Alzheimer's disease (AD), in aging, and under conditions of oxidative stress, the levels of reactive carbonyl compounds continuously increase. Accumulating carbonyl levels might be caused by an impaired enzymatic detoxification system. The major dicarbonyl detoxifying system is the glyoxalase system, which removes methylglyoxal in order to minimize cellular impairment. Although a reduced activity of glyoxalase I was evident in aging brains, it is not known how raising the intracellular methylglyoxal level influences neuronal function and the phosphorylation pattern of tau protein, which is known to be abnormally hyperphosphorylated in AD. To simulate a reduced glyoxalase I activity, we applied an inhibitor of glyoxalase I, p-bromobenzylglutathione cyclopentyl diester (pBrBzGSCp(2)), to SH-SY5Y neuroblastoma cells to induce chronically elevated methylglyoxal concentrations. We have shown that 10 microM pBrBzGSCp(2) leads to a fourfold elevation of the methylglyoxal level after 24 hr. In addition, glyoxalase I inhibition leads to reduced cell viability, strongly retracted neuritis, increase in [Ca(2+)](i), and activation of caspase-3. However, pBrBzGSCp(2) did not lead to tau "hyper"-phosphorylation despite activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase but rather activated protein phosphatases 2 and induced tau dephosphorylation at the Ser(202)/Thr(205) and Ser(396)/Ser(404) epitopes. Preincubation with the carbonyl scavenger aminoguanidine prevented tau dephosphorylation, indicating the specific effect of methylglyoxal. Also, pretreatment with the inhibitor okadaic acid prevented tau dephosphorylation, indicating that methylglyoxal activates PP-2A. In summary, our data suggest that a reduced glyoxalase I activity mimics some changes associated with neurodegeneration, such as neurite retraction and apoptotic cell death.