okadaic-acid and Glioma

Topics: Animals; Brain; Ceramides; Enzyme Activation; Ethers, Cyclic; Glioma; Humans; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2

Our previous study demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) opening via the RhoA/Rho kinase/protein kinase C (PKC)-α/β signaling pathway and that PKC-ζ is involved in this process via other mechanisms. In the present study, using an in vitro BTB model, we detected the exact signaling mechanisms by which PKC-ζ activation affects EMAP-II-induced BTB hyperpermeability. Our results showed that three types of serine/threonine (Ser/Thr) protein phosphatases (PPs), namely PP1, PP2A, and PP2B, were expressed by rat brain microvascular endothelial cells (RBMECs). There was an interaction between PKC-ζ and PP2A in RBMECs. In addition, EMAP-II induced a significant increase in both the expression and the activity of PP2A in RBMECs. Inhibition of PKC-ζ with PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI) completely blocked EMAP-II-induced PP2A activation. Conversely, inhibition of PP2A with okadaic acid (OA) had no effect on EMAP-II-induced PKC-ζ activation. Like PKC-ζ-PI, OA partially prevented EMAP-II-induced BTB hyperpermeability and occludin redistribution in RBMECs. Neither PKC-ζ-PI nor OA affected EMAP-II-induced phosphorylation of myosin light chain and redistribution of actin cytoskeleton in RBMECs. Taken together, our present study demonstrated that low-dose EMAP-II increases BTB permeability by activating the PKC-ζ/PP2A signaling pathway, which consequently leads to the disruption of TJs and impairment of endothelial barrier function.

Topics: Actin Cytoskeleton; Animals; Antineoplastic Agents; Brain Neoplasms; Cytokines; Electric Impedance; Endothelial Cells; Enzyme Inhibitors; Glioma; Myosin Light Chains; Neoplasm Proteins; Occludin; Okadaic Acid; Permeability; Phosphorylation; Protein Binding; Protein Kinase C; Protein Phosphatase 1; Protein Phosphatase 2; Rats; Rats, Wistar; RNA-Binding Proteins; Signal Transduction; Tight Junctions; Tumor Cells, Cultured

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α' or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2-PP2A pathway.

Topics: Animals; bcl-X Protein; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cyclin D1; Glioma; HEK293 Cells; Humans; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; Serine; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transplantation, Heterologous

It is becoming increasingly clear that protein phosphatases are important modulators of cellular function and that disruption of these proteins are involved in neurodegenerative disease processes. Serine/threonine protein phosphatases (PP) such as protein phosphatase PP1, PP2A, and calcineurin are involved in hyperphosphorylation of tau- as well as beta-amyloid-induced cell death. We have previously shown serine/threonine protein phosphatases to be involved in estrogen-mediated neuroprotection. The purpose of this study was to delineate the role of PP1, PP2A, and calcineurin in the mechanism of estrogen mediated neuroprotection against oxidative stress and excitotoxicity. Treatment with protein phosphatases inhibitor II, endothall, or cyclosporin A, which are specific inhibitors of PP1, PP2A, and calcineurin, respectively, did not have an effect on cell viability. However, in combination, these inhibitors adversely affected cell survival, which suggests the importance of serine/threonine protein phosphatases in maintenance of cellular function. Inhibitors of PP1, PP2A, and calcineurin attenuated the protective effects of estrogen against glutamate-induced -neurotoxicity but did not completely abrogate the estrogen-mediated protection. The attenuation of estrogen-induced neuroprotection was achieved through decrease in the activity of theses serine/threonine phosphatases without the concomitant decrease in protein expression. In an animal model, transient middle cerebral artery occlusion caused a 50% decrease in levels of PP1, PP2A, and PP2B ipsilateral to the lesion in a manner that was prevented by estradiol pretreatment. Therefore, we conclude that in the face of cytotoxic challenges in vitro and in vivo, estrogens maintain the function of PP1, PP2A, and calcineurin.

Topics: Animals; Brain Neoplasms; Calcineurin; Calcineurin Inhibitors; Cell Line, Tumor; Cell Survival; Cyclosporine; Dicarboxylic Acids; Drug Interactions; Enzyme Inhibitors; Estrogens; Glioma; Glutamic Acid; Mice; Neurons; Neuroprotective Agents; Neurotoxins; Okadaic Acid; Oxidative Stress; Protein Phosphatase 1; Protein Phosphatase 2; Rats; Stroke

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Topics: Acrylamides; Animals; Cell Line, Tumor; Cnidarian Venoms; Dose-Response Relationship, Drug; Furans; Glioma; Hybrid Cells; Kainic Acid; Macrolides; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxocins; Pyrans; Saxitoxin; Time Factors; Toxicity Tests

Okadaic acid (OA), a potent protein phosphatase inhibitor, has been known to induce apoptosis in a variety of cell types. The authors attempted to characterize further this model by identifying proteins involved in this form of programmed cell death.. Cellular proliferation was assessed using a colorimetric nonradioactive proliferation assay and cell counts. Apoptosis was determined by fluorescent microscopy. Activation of the mitogen-activated protein kinase (MAPK) pathways was determined by immunoprecipitation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38 followed by in vitro kinase assays. Western blot analyses were conducted to show inhibitory-kappaB (IkappaB) phosphorylation and degradation as well as Bax upregulation. The binding of nuclear factor-kappaB (NFkappaB) was shown by electrophoretic mobility shift assay. Okadaic acid induced cell death in T98G human malignant cell lines (50% inhibiting concentration = 20-25 nM). In T98G cells YO-PRO fluorescent staining was identified, thus indicating an apoptotic mechanism with a smaller percentage of cells undergoing necrotic cell death. Additionally OA induced JNK and MAPK activities in a time-dependent manner, increased the expression of Bax, and increased IkappaB phosphorylation and NFkappaB activation. There was a temporal correlation between these subcellular events and the detection of apoptosis morphology in glioma cells.. The authors believe that OA acts by blocking dephosphorylation events, thus activating apoptotic pathways through ERK and JNK activity. Additionally Bax, IkappaB and NFkappaB may also play a role in regulating these pathways.

Topics: Active Transport, Cell Nucleus; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Brain Neoplasms; Cell Count; Cell Division; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Microscopy, Fluorescence; Neoplasm Proteins; NF-kappa B; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction

To study the regulatory effect and possible mechanism of Tiaoxin Recipe (TXR) on animal's Alzheimer disease related tau protein phosphorylation.. NG108 cell model was treated with Okadaic acid and related parameters were determined using MTT staining, immunoblot, coimmunoprecipitation assay, etc.. Shown by MTT staining, NG108 cell activity decreased significantly after treated with Okadaic acid for 12 hrs, which could be ameliorated by TXR rat serum. Revealed by immunoblot method, the Okadaic acid induced elevation of phosphorylated tau protein could partly be reversed after co-treated with TXR rat serum. TXR extract could inhibit the binding of tau protein with presenilin-1, which may regulate the tau protein phosphorylation, and could be observed by coimmunoprocipitation.. TXR could inhibit tau protein hyperphosphorylation, which might partially be due to the TXR caused binding of presenilin-1 with tau protein.

Topics: Alzheimer Disease; Animals; Drugs, Chinese Herbal; Glioma; Male; Membrane Proteins; Mice; Neuroblastoma; Okadaic Acid; Phosphorylation; Phytotherapy; Plasma; Presenilin-1; Rats; tau Proteins; Tumor Cells, Cultured

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.

Topics: Amino Acid Sequence; Animals; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Glioma; Hybrid Cells; Molecular Sequence Data; Neuroblastoma; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Okadaic Acid; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Serine

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide found mainly in the brain, pituitary, and circulation. It inhibits most forms of inflammation by a mechanism that is not known. As most types of inflammation require activation of NF-kappa B, we investigated the effect of alpha-MSH on the activation of this transcription factor by a wide variety of inflammatory stimuli. Electrophoretic mobility shift assay showed that alpha-MSH completely abolished TNF-mediated NF-kappa B activation in a dose- and time-dependent manner. It also suppressed NF-kappa B activation induced by LPS, okadaic acid, and ceramide. The effect was specific, as the activation of the transcription factor activating protein-1 by TNF was unaffected. Western blot analysis revealed that TNF-dependent degradation of the inhibitory subunit of NF-kappa B, I kappa B alpha, and nuclear translocation of the p65 subunit of NF-kappa B were also inhibited. This correlated with suppression of NF-kappa B-dependent reporter gene expression induced by TNF. The inhibitory effect of alpha-MSH appeared to be mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Similarly, addition of membrane-permeable dibutyryl cAMP, like alpha-MSH, suppressed TNF-induced NF-kappa B activation. Overall, our results suggest that alpha-MSH suppresses NF-kappa B activated by various inflammatory agents and that this mechanism probably contributes to its anti-inflammatory effects.

Topics: alpha-MSH; Anti-Inflammatory Agents; Biological Transport; Cell Line; Cell Nucleus; Ceramides; Chloramphenicol O-Acetyltransferase; Cyclic AMP; Epithelial Cells; Gene Expression Regulation; Genes, Reporter; Glioma; HeLa Cells; Humans; Inflammation; Jurkat Cells; Lipopolysaccharides; NF-kappa B; NF-kappa B p50 Subunit; Okadaic Acid; Transcription Factor AP-1; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.

Topics: Animals; Bradykinin; Calcium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glioma; Hybrid Cells; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxazoles; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction; Thapsigargin; Type C Phospholipases

Valproic acid (VPA), a simple branched fatty acid anticonvulsant, has been demonstrated to have clinical efficacy in the treatment of manic-depressive illness (Bowden et al., 1994), but the mechanism(s) by which VPA produces its therapeutic effects remain to be elucidated. VPA's clinical antimanic action require a lag period for onset and are not immediately reversed upon discontinuation of treatment, effects that suggest alterations at the genomic level; we therefore investigated the effects of VPA on the modulation of the DNA binding activity of key transcription factors. DNA binding activities of activator protein 1 (AP-1) and cAMP responsive element binding protein (CREB) were studied in acute (hours) and chronic (days) VPA-treated rat C6 glioma cells. VPA did not affect CREB DNA binding activity, but concentration- and time-dependently increased AP-1 DNA binding activity. The activity was raised at 2 hours (the shortest time examined) and remained high after 6 days (the longest time used) of continuing VPA treatment. VPA also enhanced AP-1 DNA binding activity in human neuroblastoma (SH-SY5Y) cells. Because the effects of VPA were markedly inhibited by cycloheximide, they appear to require new protein synthesis. Taken together, the data suggest that antimanic agents may affect gene expression by modulation of the activity of major transcription factors; in view of the key roles of these nuclear transcription regulatory factors in long-term neuronal plasticity and cellular responsiveness, these effects may play a major role in VPA's therapeutic efficacy and are worthy of further study.

Topics: Activating Transcription Factor 2; Animals; Anticonvulsants; Brain; Cyclic AMP Response Element-Binding Protein; Cycloheximide; DNA, Neoplasm; Dose-Response Relationship, Drug; Glioma; Humans; Indoles; Maleimides; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Protein Synthesis Inhibitors; Rats; Time Factors; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured; Valproic Acid

C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na(+)-dependent L-[3H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated L-[3H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, D-[3H] aspartate to the same extent. In parallel assays, PMA did not, however, increase Na(+)-dependent glycine transport activity in C6 glioma. The inactive phorbol ester alpha-phorbol 12,13- didecanoate, did not stimulate L-[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na(+)/H+ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na(+)-dependent L-[3H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.

Topics: Adenylyl Cyclases; Amiloride; Amino Acid Transport System X-AG; Animals; Aspartic Acid; ATP-Binding Cassette Transporters; Calcimycin; Carrier Proteins; Colforsin; Enzyme Inhibitors; Ethers, Cyclic; Excitatory Amino Acid Transporter 3; Glioma; Glutamate Plasma Membrane Transport Proteins; Glutamates; Glycine; Kinetics; Okadaic Acid; Phosphoric Monoester Hydrolases; Protein Kinase C; Rats; Sodium; Sodium-Potassium-Exchanging ATPase; Symporters; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Sequence; Animals; Brain Neoplasms; Calcineurin; Calmodulin-Binding Proteins; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Glioma; Half-Life; Molecular Sequence Data; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Protein Serine-Threonine Kinases; Rats; Receptors, Serotonin; Staurosporine; Tumor Cells, Cultured

The expression of alphaB crystallin, hsp27, and hsp70 in C6 cells increased when they were exposed to arsenite (50 microM for 1 h) or heat (42 degrees C for 30 min), as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. When cells were exposed to arsenite in the presence of 0.1 microM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or 0.2 microM okadaic acid, an inhibitor of phosphoserine/phosphothreonine protein phosphatases, expression of alphaB crystallin was markedly enhanced. The induction of hsp27 and hosp70 expression was also stimulated to a considerable extent in the same cells. The stimulatory effect of PMA was further enhanced in the presence of okadaic acid, but it was strongly inhibited in the presence of 0.5 microM staurosporine, an inhibitor of protein kinase C. PMA and okadaic acid also stimulated the response to heat stress of the expression of alphaB crystallin, but they barely stimulated the response to heat stress of hsp27. The extent of stimulation of the arsenite-induced responses by PMA and okadaic acid was greater when the concentration of arsenite (i.e. the magnitude of the stress) was relatively low (25-50 microM). The arsenite-induced release of arachidonic acid from cells was also stimulated in the presence of PMA and/or akadaic acid, and the stimulatory effects of PMA and okadaic acid on the arsenite-induced accumulation of alphaB crystallin and hsp27 were strongly suppressed by quinacrine, an inhibitor of phospholipase A2. These results suggest that the stimulatory effects of PMA and okadaicacid on the stress responses are cuased, in part, by the increased metabolic activity of the arachidonic acid cascade, as a consequence of the activation of phospholipase A.

Topics: Alkaloids; Animals; Arachidonic Acid; Arsenites; Blotting, Northern; Blotting, Western; Enzyme Activation; Ethers, Cyclic; Glioma; Heat-Shock Proteins; Okadaic Acid; Protein Kinase C; Rats; RNA, Messenger; Serine Proteinase Inhibitors; Staurosporine; Stimulation, Chemical; Stress, Physiological; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Cross-regulation from the stimulatory phospholipase C to the adenylyl cyclase pathways was explored in neuroblastoma-glioma NG-108-15 cells in culture. Activation of protein kinase C by phorbol myristic acid resulted in a markedly attenuated activation of the inhibitory adenylyl cyclase response to delta-opiate agonists and epinephrine but not to the muscarinic agonist carbachol. The ability of okadaic acid to mimic the effects of phorbol myristic acid on the inhibitory response suggested a role for protein phosphorylation. Adenylyl cyclase activity from cells in which protein kinase C had been activated demonstrated a loss in the inhibitory adenylyl cyclase response at the level of the G-protein. Activation of protein kinase C prompted a 2-4-fold increase in phosphorylation of G1 alpha 2 in cells metabolically labeled with [32P]orthophosphate. The phosphate content of Gi alpha 2 was determined to be approximately 0.5 mol/mol subunit in the unstimulated cells and approximately 1.5 mol/mol subunit for cells in which protein kinase C was activated. The effects of okadaic acid, 4-alpha-phorbol, and calphostin C on inhibition of adenylyl cyclase in cells treated with phorbol myristic acid correlate with the effects of these agents on phosphorylation of Gi alpha 2. The time courses for attenuation of inhibitory adenylyl cyclase and that for phosphorylation of Gi alpha 2 were similar in cells challenged with phorbol myristic acid. These data argue for cross-regulation from the stimulatory protein kinase C to inhibitory adenylyl cyclase pathways at the level of Gi alpha 2 via protein phosphorylation.

Topics: Adenylyl Cyclases; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Ethers, Cyclic; Glioma; GTP-Binding Proteins; Hybrid Cells; Naphthalenes; Neuroblastoma; Okadaic Acid; Phorbols; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Rat C6 glioma cells undergo regulatory volume decrease (RVD) following hypoosmotic exposure. RVD was inhibited by the K+ channel blockers barium (10 mM) and quinine (1 mM). The mechanism of activation of the volume regulatory process was studied. Volume regulation was not observed following incubation of cells in Ca(2+)-free medium. Fluorescent measurement of intracellular free Ca2+ revealed no change following hypoosmotic exposure. Okadaic acid, an inhibitor of protein phosphatase type 1 and 2A inhibited VRD in C6 glioma cells. These results suggest that hypoosmotic RVD in C6 glioma cells involves a loss of K+ (and anion) from the cell. The activation of K+ loss is dependent on the presence of extracellular calcium (but not an increase in intracellular free calcium); and on protein dephosphorylation, either of a transport protein or another protein in the signalling pathway.

Topics: Animals; Barium; Calcium; Calcium Channels; Cell Line; Cell Size; Ethers, Cyclic; Glioma; Okadaic Acid; Osmotic Pressure; Phosphorylation; Potassium; Potassium Channel Blockers; Quinine; Rats

Okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, has been widely used as a tool for unravelling the regulation of cellular metabolic processes involving protein phosphorylation/dephosphorylation. It has recently been found that OA can induce reversible hyperphosphorylation of vimentin and reorganization of intermediate filaments [Lee et al., J. Cell. Biochem. 49: 378-393, 1992]. We report here that OA specifically induced the synthesis of a 78-kDa protein, which was identified as the 78-kDa glucose-regulated protein (GRP78) by two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis and peptide mapping. The induction of GRP78 by OA was dose-dependent and reversible. For 7 h treatments, GRP78 synthesis was initially enhanced under 50 nM OA and became the highest (about 6-fold) under 200 nM OA. Meanwhile, under 200 nM OA, GRP78 synthesis was initially enhanced after 4 h and reached its maximal level (about 8-fold) after 15 h of treatment. Subsequently, upon removal of OA, the level of OA-induced GRP78 was reduced to basal level after 12 h of recovery. Induction of GRP78 synthesis by OA was abolished in cells pretreated with actinomycin D and cycloheximide, indicating that it was regulated at the transcriptional level and its induction required de novo protein synthesis. Furthermore, OA suppressed protein glycosylation, and the result lent support to the hypothesis that suppression of protein glycosylation may correlate with induction of GRP78 synthesis.

Topics: Animals; Brain Neoplasms; Calcimycin; Carrier Proteins; Cycloheximide; Dactinomycin; Endoplasmic Reticulum Chaperone BiP; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; Glioma; Glycosylation; Heat-Shock Proteins; Molecular Chaperones; Neoplasm Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Protein Processing, Post-Translational; Rats; Tumor Cells, Cultured

The major component of amyloid plaque cores and cerebrovascular amyloid deposits found in Alzheimer disease is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence suggests that abnormalities in beta/A4 peptide production or beta/A4 peptide aggregation may underlie cerebral amyloidosis. In the present study, treatment of cells with phorbol dibutyrate, which activates protein kinase C, and/or okadaic acid, which inhibits protein phosphatases 1 and 2A, reduced beta/A4 peptide production by 50-80%. These effects were observed with APP695 and APP751 expressed in stably transfected CHO cells, as well as with endogenous APP in human glioma (Hs 683) cells. Phorbol dibutyrate also decreased beta/A4 peptide production in cells expressing various mutant forms of APP associated with familial Alzheimer disease, one of which was reported to manifest greatly increased beta/A4 peptide production in cultured cells. Mastoparan and mastoparan X, compounds which can activate phospholipase C and hence protein kinase C, also decreased beta/A4 peptide production in CHO cells stably transfected with APP695. A model is presented in which decreases in beta/A4 peptide production can be achieved by accelerating the metabolism of APP through a nonamyloidgenic secretory pathway.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Animals; CHO Cells; Cricetinae; Ethers, Cyclic; Glioma; Humans; Kinetics; Mutagenesis, Site-Directed; Okadaic Acid; Phorbol 12,13-Dibutyrate; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Recombinant Proteins; Transfection; Tumor Cells, Cultured

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.

Topics: Animals; beta-Galactoside alpha-2,3-Sialyltransferase; Calmodulin; Carbohydrate Sequence; Ethers, Cyclic; Glioma; Glycosylation; Homeostasis; Imidazoles; Kinetics; Marine Toxins; Molecular Sequence Data; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Sialyltransferases; Sulfonamides; Tumor Cells, Cultured

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108-15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32P]-ortho-phosphate, laminin caused a decrease in the TCA-precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin-mediated process formation. Inhibitors of kinase activity such as H-7, H-8, and H-9 increased laminin-mediated neural process formation. Since phosphate incorporation into laminin-binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin-mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.

Topics: Adrenal Gland Neoplasms; Animals; Cell Adhesion; Diglycerides; Ethers, Cyclic; Glioma; Laminin; Neoplasm Proteins; Neuroblastoma; Neurons; Okadaic Acid; Pheochromocytoma; Phorbol Esters; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Tumor Cells, Cultured; Vanadates