We investigated the role of Ca2+ and protein kinases/phosphatases in the stimulatory effect of insulin on glucose transport. In isolated rat adipocytes, the simple omission of CaCl2 from the incubation medium significantly reduced, but did not abolish, insulin-stimulated 2-deoxy glucose (2-DG) uptake. Pre-loading adipocytes with intracellular Ca2+ chelator, 5,5'-dimethyl bis (o-aminophenoxy)ethane-N,N,N'N' tetraacetic acetoxymethyl ester (5,5'-dimethyl BAPTA/AM) completely blocked the stimulation. Insulin raised intracellular Ca2+ concentration ((Ca2+)i) about 1.7 times the basal level of 72+/-5 nM, and 5,5'-dimethyl BAPTA/AM kept it constant at the basal level. This correlation between insulin-induced increases in 2-DG uptake and (Ca2+)i indicates that the elevation of (Ca2+)i may be prerequisite for the stimulation of glucose transport. Studies with inhibitors (ML-9, KN-62, cyclosporin A) of Ca2+-calmodulin dependent protein kinases/phosphatases also indicate an involvement of intracellular Ca2+. Additional studies with okadaic acid and calyculin A, protein phosphatase-1 (PP-1) and 2A (PP-2A) inhibitors, indicate an involvement of PP-1 in insulin action on 2-DG uptake. These results indicate an involvement of Ca2+-dependent signaling pathway in insulin action on glucose transport.
Adipocytes* ; Animals ; Cyclosporine ; Glucose* ; Insulin* ; Okadaic Acid ; Rats ; Staphylococcal Protein A

To understand the cytotoxic mechanism of MPP+, we examined the involvement of ceramide in MPP+ -induced cytotoxicity to human neuroblastoma SH-SY5Y cells. When SH-SY5Y cells were exposed to MPP+, MPP+ induced dose-dependent cytotoxicity accompanied by 2-fold elevation of intracellular ceramide levels in SH-SY5Y cells. Three methods were used to test the hypothesis that the elevated intracellular ceramide is related to MPP+ -induced cytotoxicity: C2-ceramide was directly applied to cells, sphingomyelinase (SMase) was exogenously added, and oleoylethanolamine (OE) was used to inhibit degradation of ceramide. Furthermore, inhibition of ceramide-activated protein phosphatase (CAPP), the effector of ceramide, using okadaic acid (OA) attenuated cell death but treatment of fumonisin B1, the ceramide synthase inhibitor, did not alter the cytotoxic effect of MPP+. Based on these, we suggest that the elevation of intracellular ceramide is one of the important mediators in MPP+ -induced cell death.
Cell Death ; Humans* ; Neuroblastoma* ; Okadaic Acid ; Sphingomyelin Phosphodiesterase

Rats ; Animals ; Okadaic Acid/toxicity* ; PC12 Cells ; Triterpenes ; tau Proteins/metabolism* ; Phosphorylation

OBJECTIVETo investigate the effect of acteoside on SK-N-SH nerve cell injury induced by okadaic acid (OA).

METHODSK-N-SH nerve cells were processed with 20 nmol * L OA to establish the Alzheimer's disease (AD) cellular model, and 5, 10, 20 mg . L-1 acteoside was used to antagonize against its effect. Cell morphology was observed under inverted microscope. The cell survival rate was detected with MTT, and the LDH release rate was measured by enzyme label kit. Western blot was applied to determine the expression of phosphorylation tau proteins in nerve cells.

RESULTThe acteoside could significantly improve SK-N-SH cell morphology, enhance the cell survival rate, decrease the cell LDH release rate and the expression of phosphorylated tau proteins at p-Ser 199/202 and p-Ser 404 sites, up-regulated the expression of at non-phosphorylated tau proteins at Ser 202 site and Ser 404 sites.

CONCLUSIONActeoside has significant protective effect on nerve cell injury induced by OA.

Alzheimer Disease ; metabolism ; Cell Line ; Cell Survival ; drug effects ; Glucosides ; pharmacology ; Humans ; Okadaic Acid ; Phenols ; pharmacology ; tau Proteins ; metabolism

Several signal transduction pathways have been implicated in ischemic preconditioning induced by the activation of ATP-sensitive K+ (KATP) channels. We examined whether protein kinase C (PKC) modulated the activity of KATP channels by recording KATP channel currents in rabbit ventricular myocytes using patch-clamp technique and found that phorbol 12, 13-didecanoate (PDD) enhanced pinacidil-induced KATP channel activity in the cell-attached configuration; and this effect was prevented by bisindolylmaleimide (BIM). KATP channel activity was not increased by 4alpha-PDD. In excised inside-out patches, PKC stimulated KATP channels in the presence of 1 mM ATP, and this effect was abolished in the presence of BIM. Heat-inactivated PKC had no effect on channel activity. PKC-induced activation of KATP channels was reversed by PP2A, and this effect was not detected in the presence of okadaic acid. These results suggest that PKC activates KATP channels in rabbit ventricular myocytes.
Adenosine Triphosphate ; Ischemic Preconditioning ; KATP Channels* ; Muscle Cells* ; Okadaic Acid ; Patch-Clamp Techniques ; Protein Kinase C* ; Protein Kinases* ; Signal Transduction

The T cell antigen receptor (TcR) in combination with costimulatory signals triggered by accessory molecules present on the surface of the antigen-presenting cells (APC) regulates the activation and growth of T lymphocytes. Calyculin A and Okadaic acid is known to be an inhibitor of serine/threonine phosphatase and RK-682 specifically blocks functions of tyrosine phosphatase. To investigate roles of these inhibitors in TcR-mediated signaling cascade, chimeric molecule CD8-5 which contains the extracellular and transmembrane domains of the human CD8a molecule and the cytoplasmic tail of TcR 5 chain were stably expressed in Jurkat cell line. CD8-5 chimeric protein induced tyrosine phosphorylation of various cytoplasmic substrates and IL-2 gene expression in a NFAT dependent manner by stimulation with anti-CD8 mAb OKT8 as seen in TcR stimulation. When CD8-5 transfectants were preincubated with Okadaic acid, Calyculin or RK682, they differentially affected tyrosine phosphorylation of signaling mediators including CD8-5 molecule. When Jurkat Tag cell line was used where SV40 T antigen is stably expressed and the expression of p-galactosidase is driven by the multiple NFAT binding sites plus minimal IL-2 promoter, these phosphatase inhibitors -RK682, Calyculin A, Okadaic acid- effectively inhibited IL-2 gene expression at the concentration of 1.2832 x 10 ' M, 3.9924 x 10 M, 7.2707 x 10 M respectively. These results suggested that Okadaic acid, Calyculin or RK682 modulate TcR-proximal as well as TcR-distal signaling events during T cell activation.
Antigen-Presenting Cells ; Antigens, Viral, Tumor ; Binding Sites ; Cell Line ; Cytoplasm ; Gene Expression ; Humans ; Interleukin-2 ; Jurkat Cells ; Okadaic Acid* ; Phosphorylation ; Receptors, Antigen, T-Cell ; T-Lymphocytes ; Tyrosine

OBJECTIVETo study the changes in expression and activity of protein phosphatases type 2A (PP2A ) during differentiation of NB4 and NB4-MR2 cells induced by all-trans retinoic acid (ATRA), and evaluate the role of PP2A in MR2 resistance to ATRA.

METHODSATRA, okadaic acid (OKA) and ATRA + OKA at the same dosage were incubated with NB4 and MR2 cells respectively. Wright's staining and NBT reduction test were employed to evaluate the change in the cells. The CD11b expression was measured by flow cytometry. The activity of PP2A was evaluated by serine/threonine phosphatase assay system, and the level of PP2A subunits was detected by Western blot.

RESULTS1) Wright's staining, NBT reduction test and flow cytometry results showed OKA could augment the differentiation of NB4 induced by ATRA, and OKA + ATRA induced slight differentiation of MR2 cells. 2) Phosphatase assay showed a decrease in PP2A phosphatase activity [(534 +/- 43) pmol x min(-1) x microg protein(-1)] in NB4 after ATRA treatment, accompanied with that activity [(959 +/- 83) pmol x min(-1) x microg protein(-1)] in untreated NB4 cells. OKA enhanced the inhibitory effect of ATRA on the activity in NB4. When OKA + ATRA was incubated with MR2, PP2A in the cells was significantly decreased [(229 +/- 23) pmol x min(-1) x microg protein(-1)]. 3) Western blot analysis showed that the level of PP2A catalytic subunit (PP2A/C) was decreased during the course of ATRA-induced NB4 cell differentiation, whereas expressions of every subunits of PP2A in MR2 cells were somewhat unaltered.

CONCLUSIONExpression of PP2A/C and activity of PP2A is decreased during differentiation of NB4 induced by ATRA, and no repression of the PP2 activity maybe related to MR2 resistance to ATRA.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Okadaic Acid ; pharmacology ; Phosphoprotein Phosphatases ; metabolism ; Protein Phosphatase 2 ; antagonists & inhibitors ; metabolism ; Tretinoin ; pharmacology

OBJECTIVETo investigate the effects of insulin-like growth factor-1 (IGF-1) on cell injuries and tau hyperphosphorylation induced by okadaic acid (OA).

METHODSThe experimental groups were designed as follows: (1) SH-SY5Y culture (control group); (2) SH-SY5Y exposed to 40 nmol/L OA for 24 hours (OA group); (3) SH-SY5Y exposed to OA for 24 hours in the presence of 2 hour pretreatment with 100, 200 and 400 ng/ml IGF-1 (IGF-1 pretreatment groups). The changes of cell morphology were observed by inverted microscope. The viability of cells was detected by MTT. The injuries of cells were examined by Hoechst 33258 staining and the activity of caspase-3. Western-blot was applied to determine the expression of phosphorylation of tau protein.

RESULTSIn IGF-1 pretreatment group, the cell morphology was improved, the viability of cells was increased, and caspase-3 activation and hyperphosphorylation of tau (Ser396) were reduced.

CONCLUSIONIGF-1 can protect the SH-SY5Y cells from cell injuries induced by OA by inhibiting tau hyperphosphorylation.

Cell Line, Tumor ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Neuroblastoma ; pathology ; Neuroprotective Agents ; pharmacology ; Okadaic Acid ; antagonists & inhibitors ; toxicity ; Phosphorylation ; drug effects ; tau Proteins ; chemistry

The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of Ca2+-activated potassium channel (KCa channel) and is involved in the activation of KCa channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1, 50 micrometer) and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP, 100 micrometer), a membrane-permeable analogue of cGMP, increased the KCa channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP (100 micrometer), cGMP (100 micrometer), ATP+cGMP (100 micrometer each), PKG (5 U/ microliter), ATP (100 micrometer)+PKG (5 U/ microliter), or cGMP (100 micrometer)+PKG (5 U/ microliter) did not increase the channel activity. ATP (100 micrometer)+cGMP (100 micrometer)+PKG (5 U/ microliter) added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP, 100 micrometer), a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The KCa channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of KCa channels, or some associated proteins in the membrane patch increase the activity of the KCa channel, and the activation may be associated with the vasodilating action.
Adenosine Triphosphate ; Cyclic GMP-Dependent Protein Kinases ; Membranes ; Molsidomine ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Okadaic Acid ; Patch-Clamp Techniques ; Phosphorylation ; Potassium Channels* ; Potassium* ; Protein Phosphatase 2 ; Signal Transduction*

To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 microl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno positive reaction for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated.
Animals ; Astrocytes ; drug effects ; metabolism ; Axons ; drug effects ; metabolism ; Brain ; cytology ; Dendrites ; drug effects ; metabolism ; Excitatory Amino Acid Transporter 1 ; metabolism ; Neurofibrillary Tangles ; pathology ; Neurons ; drug effects ; metabolism ; Okadaic Acid ; pharmacology ; Phosphorylation ; Rats ; tau Proteins ; metabolism