Telmisartan Inhibits Nitric Oxide Production and Vessel Relaxation via Protein Phosphatase 2A-mediated Endothelial NO Synthase-Ser¹¹⁷⁹ Dephosphorylation

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser¹¹⁷⁹ (p-eNOS-Ser¹¹⁷⁹). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser¹¹⁷⁹ levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser¹¹⁷⁹ levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser¹¹⁷⁹ levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser¹¹⁷⁹ dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.

Study on protective effect of acteoside on cellular model of Alzheimer's disease induced by okadaic acid / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of acteoside on SK-N-SH nerve cell injury induced by okadaic acid (OA).</p><p><b>METHOD</b>SK-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.</p><p><b>RESULT</b>The 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.</p><p><b>CONCLUSION</b>Acteoside has significant protective effect on nerve cell injury induced by OA.</p>

Influence of PP2A activator on proliferation of HL-60 cells and analysis of PP2A activity changes in patients with acute myeloid leukemia / 中国实验血液学杂志

In order to investigate the effect of PP2A activator and PP2A inhibitor on proliferation of HL-60 cells and analyze the changes of PP2A activity in patients with acute myeloid leukemia (AML), HL-60 cells were treated with FTY720 alone or in combination with okadaic acid (OA) for 24 hours in culture. Cell proliferation was assayed with CCK8 kit. In addition, 20 AML patients including de novo AML and relapsed AML were enrolled in this study. The activity of PP2A in the peripheral blood mononuclear cells of patients was assayed with a PP2A Immunoprecipitation Phosphatase Assay Kit, the data were analyzed by software SPSS 16.0. The results indicated that as compared with control group, the proliferation of cells in FTY720 group was obviously inhibited (p < 0.05). The proliferation of cells in FTY720 + OA group was slightly inhibited as compared with the control group, there was no statistical difference (p > 0.05), but there was significant difference between the FTY720 + OA and FTY720 groups (p < 0.05). The activity of PP2A in AML patients (453.67 ± 102.52 pmol phosphate) was obviously lower than that in the normal controls (673.29 ± 96.32 pmol phosphate), there was significant difference between them (p < 0.01). It is concluded that the activation or inhibition of PP2A can affect the proliferation of HL-60 cells in vitro. Compared with healthy individuals, the activity of PP2A in AML patients is obviously lower. PP2A protein playing a key role in the occurrence and development of AML may be valuable for the diagnosis and treatment of AML.

Effects of intrahippocampal administration of the phosphatase inhibitor okadaic acid: dual effects on memory formation / Efeitos da administração intra-hipocampal do inibidor de fosfatases ácido okadaico: efeito duplo sobre a formação de memória

Protein phosphorylation mediated by serine-threonine kinases in the hippocampus is crucial to the synaptic modifications believed to underlie memory formation. The role of phosphatases has been the focus of comparatively little study. Objectives: Here we evaluate the contribution of the serine-threonine protein phosphatases 1 and 2A (PP1, PP2A) on memory consolidation. Methods: We used immediate post-training bilateral hippocampal infusions of okadaic acid (OA, 0.01 and 10 pmol/side), a potent inhibitor of PP1 and PP2A, and measured short- [3 h] and long-term memory [24 h] (STM, LTM) of step-down inhibitory avoidance. Results: At the lower dose, OA inhibited both STM and LTM whereas at the higher dose it instead enhanced LTM. Pre-test infusion of these two doses of OA had no effect on retrieval. Conclusions: These two doses of OA are known to selectively inhibit PP1 and PP2A respectively. These findings point to the importance of these enzymes in memory formation and also suggest a deleterious influence of endogenous hippocampal PP2A on LTM formation.

A fosforilação de proteínas mediada por serina-treonina quinases no hipocampo é crucial para as modificações sinápticas que se acredita sejam necessárias para a formação de memórias. O papel das fosfatases tem sido comparativamente pouco estudado. Objetivos: Aqui avaliamos a contribuição das fosfatases serina-treonina 1 e 2 (PP1, PP2A) sobre a consolidação da memória. Métodos: Usamos infusões imediatamente após o treino de ácido okadaico (OA, 0.01 e 10 pmol/lado), um potente inibidor de PP1 e medimos memória de curta [3 h] e longa duração [24 h] (STM, LTM) de esquiva inibitória de evitar descer de uma plataforma. Resultados: Na dose menor, OA inibiu tanto STM como LTM. Na dose maior, produziu, em vez disso, uma melhora da LTM. A infusão pré-teste de qualquer uma das duas doses de OA não teve efeito sobre a evocação. Conclusões: Estas duas doses de OA são conhecidas por inibir seletivamente PP1 a PP2 respectivamente. Estes resultados apontam à importância das duas enzimas na formação de memória e sugerem, adicionalmente, uma influência deletérea da PP2A endógena sobre a formação de LTM.

Protective effects of IGF-1 on cell injuries and tau hyperphosphorylation induced by okadaic acid / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of insulin-like growth factor-1 (IGF-1) on cell injuries and tau hyperphosphorylation induced by okadaic acid (OA).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>IGF-1 can protect the SH-SY5Y cells from cell injuries induced by OA by inhibiting tau hyperphosphorylation.</p>

Action of protein phosphatase-1 on Tat-dependent HIV-1 transcription and its related inhibitors / 药学学报

Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.

beta-Secretase inhibitor increases amyloid-beta precursor protein level in rat brain cortical primary neurons induced by okadaic acid / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Senile plaques and neurofibrillary tangles (NFTs) represent two of the major histopathological hallmarks of Alzheimer's disease (AD). The plaques are primarily composed of aggregated amyloid beta (Abeta) peptides. The processing of amyloid-beta precursor protein (AbetaPP) in okadaic acid (OA)-induced tau phosphorylation primary neurons was studied.</p><p><b>METHODS</b>Primary cultures of rat brain cortical neurons were treated with OA and beta-secretase inhibitor. Neurons' viability was measured. AbetaPP processing was examined by immunocytochemistry and Western blotting with specific antibodies against the AbetaPP-N-terminus (NT) and AbetaPP-C-terminus (CT).</p><p><b>RESULTS</b>Ten nmol/L OA had a time-dependent suppression effect on primary neurons' viability. The suppression effect was alleviated markedly by pretreatment with beta-secretase inhibitor. After OA treatment, both AbetaPP and beta-C-terminal fragment (betaCTF) were significantly increased in neurons. AbetaPP level was increased further in neurons pretreated with beta-secretase inhibitor.</p><p><b>CONCLUSIONS</b>In OA-induced tau phosphorylation cell model, inhibition of beta-secretase may protect neurons from death induced by OA. Because of increased accumulation of AbetaPP in neurons after OA treatment, more AbetaPP turns to be cleaved by beta-secretase, producing neurotoxic betaCTF. As apotential effective therapeutic target, beta-secretase is worth investigating further.</p>

Effect of okadaic acid on differentiation of NB4 and MR2 cells induced by all-trans retinoic acid / 中华血液学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>ATRA, 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.</p><p><b>RESULTS</b>1) 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.</p><p><b>CONCLUSION</b>Expression 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.</p>

Primeira detecção de ácido ocadáico em ostras (Crassostrea rhizophorae), coletadas no canal de Santa Cruz, Itapissuma, Pernambuco / First detention of acid ocadaico in oysters (Crassostrea rhizophorae), collected in the Santa Cruz, Itapissuma, Pernambuco

O ácido ocadáico (AO) é uma importante toxina, produzida por dinoflagelados, capaz de causar no homem o envenenamento diarréico por moluscos conhecido como Diarrhetic Shellfish Poisoning (DSP). Embora uma toxina fatal, esta ficotoxina está envolvida na inibição de certas proteínas e no aparecimento de neoplasias, que o torna uma toxina perigosa. Desta forma, considerando o risco do consumo de moluscos bivalves e a escassez de pesquisas para sua detecção e quantificação no Estado de Pernambuco, foram analisados extratos preparados a partir do hepatopâncreas de ostras (Crassostrea rhizophorae) coletadas no Canal de Santa Cruz localizado no Município de Itapissuma – PE, em quatro pontos distintos.O ácido ocadáico foi analisado através da Cromatografia Líquida de Alta Eficiência com Detecção Fluorimétrica. (...) Embora as concentrações do AO encontradas tenham sido baixas, estando as ostras aptas ao consumo humano segundo limites adotados (2000ng AO.g-1 hepatopâncreas de moluscos) por vários países, há uma tendência internacional de exigir a total ausência de toxinas diarréicas devido ao seu potencial efeito carcinogênico para consumidores regulares de moluscos. A presença inédita do AO no Canal de Santa Cruz é um alerta às autoridades sanitárias para que mais pesquisas sejam realizadas na região e em outras localizadas do Estado.

Protective effect of (-) clausenamide against neurotoxicity induced by okadaic acid and beta-amyloid peptide25-35 / 药学学报

This study is to investigate the protective effect of (-) clausenamide against the neurotoxicity of okadaic acid in SH-SY5Y cell line, and injection beta-amyloid peptide25-35 (Abeta25-35) to the cerebral ventricle in ovariectomy (OVX) rats. MTT assay, LDH assay, and Hoechst 33258 staining were used to detect the effect of (-) clausenamide on the toxicity of okadaic acid in SH-SY5Y cell line. The animal model was induced by ovariectomized and injection of Abeta25-35 in the cerebroventricle of rats. The effect of (-) clausenamide on learning and memory deficiency was observed by step-through test. Electron microscope, Nissl body staining, and HE staining were used to examine the morphological changes in hippocampus and cerebral cortex neurons. Pretreatment of (-) clausenamide and LiCl decreased the rate of cell death from MTT, LDH release, and apoptosis from Hoechst 33258 staining in SH-SY5Y cell line. The step-through tests showed (-) clausenamide could improve the ability of learning and memory. The Nissl body staining and HE staining experiments also showed the neuroprotective effects of (-) clausenamide on the neurons of hippocampus and cerebral cortex. (-) Clausenamide has the protective effects against the neurotoxicity induced by okadaic acid and Abeta25-35.

Protein Kinase C Activates ATP-sensitive Potassium Channels in Rabbit Ventricular Myocytes

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.

Ginsenoside Rb1 attenuates okadaic acid-induced Tau protein hyperphosphorylation in rat hippocampal neurons / 生理学报

The present study was aimed to investigate the effects of ginsenoside Rb1 on okadaic acid (OA)-induced Tau hyperphosphorylation in hippocampal neurons of Sparague-Dawley rat and to explore its possible mechanism. Animals were randomly divided into four groups. Group 1 received dimethysulphoxide (DMSO) injection (vehicle group), group 2 only received OA injection (OA group), group 3 was pretreated with Rb1 and then received OA injection (Rb1 pretreatment group), and the group 4 was an intact control group. The animals in group 3 were injected intraperitoneally with various doses of Rb1 at 5, 10, and 20 mg/kg (once a day for 14 d). On the thirteen day of pretreatment, animals in Rb1 pretreatment group as well as animals in OA group received a bolus injection of 0.483 microg of OA (1.5 microl of solution in DMSO) at right dorsal aspect of hippocampus to induce Tau hyperphosphrylation. The brains were harvested one day after the last treatment. In all groups, the morphology of neurofibrils, phosphorylation of Tau protein, and the activity of phosphatase 2A (PP2A) were investigated. In OA group, the Bielschowski's assay revealed darkened and uneven neurofibrils staining in the hippocampus. The immunohistochemistry results showed a significant increase in Thr(231) phosphorylation of Tau protein in OA group relative to the control group (P<0.01). OA injection also markedly decreased PP2A activity (P<0.01). Western blot confirmed Thr(231) phosphorylation of Tau protein and it also detected phosphorylation of Ser(396) of Tau protein. The animals with Rb1 pretreatment displayed even staining of neurofibrils and normal pattern of fiber organization. Rb1 pretreatment also attenuated Thr(231) and Ser(396) hyperphosphorylations of Tau protein, and restored PP2A activity compared to the OA group (P<0.01). These results indicate that OA-induced hyperphosphorylation of Tau protein in rat hippocampal neurons can be attenuated by the pretreatment of ginsenoside Rb1. These data also implicate that Rb1 has potential neuroprotective effects on Tau-related neuropathology.

Environmental and health effects associated with Harmful Algal Bloom and marine algal toxins in China / 生物医学与环境科学（英文）

The frequency and scale of Harmful Algal Bloom (HAB) and marine algal toxin incidents have been increasing and spreading in the past two decades, causing damages to the marine environment and threatening human life through contaminated seafood. To better understand the effect of HAB and marine algal toxins on marine environment and human health in China, this paper overviews HAB occurrence and marine algal toxin incidents, as well as their environmental and health effects in this country. HAB has been increasing rapidly along the Chinese coast since the 1970s, and at least 512 documented HAB events have occurred from 1952 to 2002 in the Chinese mainland. It has been found that PSP and DSP toxins are distributed widely along both the northern and southern Chinese coasts. The HAB and marine algal toxin events during the 1990s in China were summarized, showing that the HAB and algal toxins resulted in great damages to local fisheries, marine culture, quality of marine environment, and human health. Therefore, to protect the coastal environment and human health, attention to HAB and marine algal toxins is urgently needed from the environmental and epidemiological view.

Citotoxicidade do promotor de tumor e sua ação mitogênica sobre os linfócitos humanos / Cytotoxicity studies of okadaic acid on human lymphocytes and its mitogenic effect

Proteínas fosfatases são moléculas sinalizadoras que agem juntamente com as proteínas quinases para regular uma variedade de processos celulares fundamentais, bem como, crescimento celular, mitogênese, metabolismo, transcrição de gene, ciclo celular e resposta ao estresse e imune. O ácido okadáico é um potente e específico inibidor de proteína serina/treonina fosfatase (PP1 e PP2A). O objetivo deste estudo foi avaliar o efeito citotóxico do ácido okadáico na viabilidade de linfócitos humanos e sua ação mitogênica. Nos estudos de citotoxicidade avaliamos o efeito do ácido okadáico através dos seguintes parâmetros: redução do MTT (integridade mitocondrial), conteúdo total de proteínas (número de células) e atividade fosfatásica (metabolismo celular). O valor para redução do MTT e atividade fosfatase foram: 50nM e 100nM, respectivamente; não foi encontrado valor de IC50 para o conteúdo de proteína. A atividade fosfatásica não foi afetada pelo ácido okadáico (100nM) quando este composto foi adicionado no extrato celular. A proliferação de linfócitos foi estimulada em 25 porcento quando as células foram tratadas com o ácido okadáico durante o plaqueamento e na ausência da fitohemaglutinina (mitogêno). O estímulo máximo foi até 30 minutos. O ácido okadáico não foi citotóxico para os linfócitos. A ação mitogênica deste composto foi confirmada pelo conteúdo de proteína.

Okadaic acid induces the expression of glutamate transporter EAAT1 in the neurons of rat brain / 生理学报

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.

Ceramide is Involved in MPP+ -induced Cytotoxicity in Human Neuroblastoma Cells

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.

Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.

Biochemical characterization and inhibitory effects of dinophysistoxin-1, okadaic acid and microcystine 1-r on protein phosphatase 2a purified from the mussel Mytilus chilensis

Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.

Modulation of Ca2+ activated potassium channels by cGMP-dependent signal transduction mechanism in cerebral arterial smooth muscle cells of the rabbit

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.

Role of Ca2+ in the stimulation of glucose transport by insulin in adipocytes

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.