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1.
Int J Obes (Lond) ; 38(8): 1044-52, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24232498

RESUMO

OBJECTIVES: Activation of the Wnt/ß-catenin signaling pathway inhibits adipogenesis by maintaining preadipocytes in an undifferentiated state. We investigated the effect of indirubin-3'-oxime (I3O), which was screened as an activator of the Wnt/ß-catenin signaling, on inhibiting the preadipocyte differentiation in vitro and in vivo. METHODS: 3T3L1 preadipocytes were differentiated with 0, 4 or 20 µM of I3O. The I3O effect on adipocyte differentiation was observed by Oil-red-O staining. Activation of Wnt/ß-catenin signaling in I3O-treated 3T3L1 cells was shown using immunocytochemical and immunoblotting analyses for ß-catenin. The regulation of adipogenic markers was analyzed via real-time reverse transcription-PCR (RT-PCR) and immunoblotting analyses. For the in vivo study, mice were divided into five different dietary groups: chow diet, high-fat diet (HFD), HFD supplemented with I3O at 5, 25 and 100 mg kg(-1). After 8 weeks, adipose and liver tissues were excised from the mice and subject to morphometry, real-time RT-PCR, immunoblotting and histological or immunohistochemical analyses. In addition, adipokine and insulin concentrations in serum of the mice were accessed by enzyme-linked immunosorbent assay. RESULTS: Using a cell-based approach to screen a library of pharmacologically active small molecules, we identified I3O as a Wnt/ß-catenin pathway activator. I3O inhibited the differentiation of 3T3-L1 cells into mature adipocytes and decreased the expression of adipocyte markers, CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ, at both mRNA and protein levels. In vivo, I3O inhibited the development of obesity in HFD-fed mice by attenuating HFD-induced body weight gain and visceral fat accumulation without showing any significant toxicity. Factors associated with metabolic disorders such as hyperlipidemia and hyperglycemia were also improved by treatment of I3O. CONCLUSION: Activation of the Wnt/ß-catenin signaling pathway can be used as a therapeutic strategy for the treatment of obesity and metabolic syndrome and implicates I3O as a candidate anti-obesity agent.


Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Indóis/farmacologia , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Oximas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Células 3T3-L1/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Dieta Hiperlipídica , Masculino , Medicina Tradicional Chinesa , Síndrome Metabólica/tratamento farmacológico , Camundongos , Obesidade/tratamento farmacológico , Reação em Cadeia da Polimerase/métodos
2.
Cell Death Differ ; 15(11): 1782-93, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18636075

RESUMO

The enzymatic activity of phospholipase D (PLD) is known to be essential for cell survival and protection from apoptosis. However, the mechanisms regulating PLD activity during apoptosis remain unknown. Here we report that cleavage of PLD1 by caspases facilitates p53-mediated apoptosis. Cleavage of PLD1 into an N-terminal fragment (NF-PLD1) and a C-terminal fragment at the amino-acid sequence, DDVD(545), led to a reduction in PLD1 activity. However, a caspase-resistant mutant form of PLD1 retained significant levels of enzymatic activity and apoptotic function as compared to wild-type PLD1. Exogenous NF-PLD1 expression induced apoptosis through a dominant-negative effect on the activity of endogenous PLD1. During apoptosis, a small fraction of PLD1 is cleaved by caspases in a p53-independent manner and NF-PLD1 amplifies apoptotic signaling through inhibition of the remaining PLD1 activity. As PLD1 suppresses the ATM-Chk2-p53 pathway, elimination of PLD1 activity through NF-PLD1 or si-RNA against PLD1 increases apoptosis in a p53-dependent manner. Taken together, our results reveal that cleavage of PLD1 by caspases promotes apoptosis via modulation of the p53-dependent cell death pathway.


Assuntos
Apoptose , Caspases/metabolismo , Fosfolipase D/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dano ao DNA , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Fosfolipase D/química , Termodinâmica
3.
FEBS Lett ; 499(1-2): 82-6, 2001 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-11418117

RESUMO

Sphingolipid metabolites, such as sphingosine and ceramide, are known to play important roles in cell proliferation, differentiation and apoptosis, but the physiological roles of phytosphingosine (PHS) and phytoceramide (PHC) are poorly understood. In this study we investigated the effects of PHS, C2-PHC (N-acetylPHS) and C6-PHC (N-hexanoylPHS) on cell growth and intracellular signalling enzymes. Treatment of Chinese hamster ovary (CHO) cells with PHS, C2-PHC or C6-PHC resulted in cell death in a time- and dose-dependent manner. C2-PHC induced internucleosomal DNA fragmentation, whereas PHS or C6-PHC had little if any effect on DNA fragmentation under the same experimental conditions. Both PHS and C2-PHC inhibited carbachol-induced activation of phospholipase D (PLD), but not of phospholipase C (PLC), in CHO cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor (mAChR). On the other hand, no significant effect of C6-PHC on PLD or PLC was observed. Our results show that PHS and C2-PHC exert strong cytotoxic effects on CHO cells and modulate the mAChR-mediated signal transduction pathway.


Assuntos
Apoptose/efeitos dos fármacos , Caenorhabditis elegans , Carbacol/farmacologia , Ceramidas/farmacologia , Fosfolipase D/metabolismo , Receptores Muscarínicos/metabolismo , Esfingosina/farmacologia , Animais , Células CHO , Divisão Celular/efeitos dos fármacos , Ceramidas/química , Ceramidas/toxicidade , Cricetinae , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Receptores Muscarínicos/genética , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/toxicidade , Fatores de Tempo , Fosfolipases Tipo C/metabolismo
4.
FEBS Lett ; 331(1-2): 38-42, 1993 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-8405407

RESUMO

A 130 kDa PLC isozyme was purified from the particulate fraction of bovine cerebellum. This PLC was recognized by a polyclonal antiserum generated against the purified 97 kDa PLC-beta 4. Reconstitution of the purified 130 kDa PLC with the membranes of C6 Bu-1 cells in the presence of GTP gamma S or AlF4- resulted in PLC activation as well as the association of PLC with the membranes. Both the association and activation were revoked when the membrane was washed with 2 M KCl. The 97 kDa PLC-beta 4 did not associate with membranes. These data suggest that the 130 kDa PLC is the intact form of PLC-beta 4 the activity of which is likely to be regulated by a G-protein on the membrane.


Assuntos
Cerebelo/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Bovinos , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Ratos , Células Tumorais Cultivadas
5.
FEBS Lett ; 358(2): 105-8, 1995 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-7828717

RESUMO

Phospholipase C (PLC)-gamma 1 and -gamma 2 play a pivotal role in signal transduction for cell proliferation and differentiation. The enzyme activity and protein level of PLC-gamma 1 were markedly decreased in the human histiocytic leukemia U937 cell line during the differentiation process which is induced by phorbol 12-myristate 13-acetate (PMA) but those of PLC-gamma 2 were not altered. Northern blot analysis showed that the levels of PLC-gamma 1 and -gamma 2 transcripts were not changed. These results suggest that the expression of PLC-gamma 1 during the PMA-induced differentiation may be down-regulated by post-transcriptional processing.


Assuntos
Diferenciação Celular , Isoenzimas/genética , Fosfolipases Tipo C/genética , Divisão Celular , Regulação para Baixo , Humanos , Isoenzimas/biossíntese , Fosfolipase C gama , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Fosfolipases Tipo C/biossíntese
6.
FEBS Lett ; 454(3): 197-200, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10431806

RESUMO

Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.


Assuntos
Fosfolipase D/biossíntese , Teratocarcinoma/enzimologia , Teratocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Regulação para Baixo/efeitos dos fármacos , Camundongos , Tretinoína/farmacologia , Células Tumorais Cultivadas
7.
FEBS Lett ; 508(2): 277-81, 2001 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-11718730

RESUMO

Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress-induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose- and time-dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan-1-ol, a PLD inhibitor, attenuated both pervanadate-induced necrosis and increase of intracellular Ca(2+). Removal of extracellular Ca(2+) inhibited pervanadate-induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate-induced necrosis of VSMCs, which is at least partly due to Ca(2+) toxicity.


Assuntos
Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Estresse Oxidativo , Fosfolipase D/metabolismo , Animais , Aorta/citologia , Butanóis/farmacologia , Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Peróxido de Hidrogênio/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Necrose , Estresse Oxidativo/efeitos dos fármacos , Ratos , Fatores de Tempo , Vanadatos/farmacologia
8.
Neuropharmacology ; 41(4): 443-53, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11543764

RESUMO

The effects of fluoxetine and its major metabolite, norfluoxetine, were studied using the patch-clamp technique on the cloned neuronal rat K(+) channel Kv3.1, expressed in Chinese hamster ovary cells. In whole-cell recordings, fluoxetine and norfluoxetine inhibited Kv3.1 currents in a reversible concentration-dependent manner, with an IC(50) value and a Hill coefficient of 13.11+/-0.91 microM and 1.33+/-0.08 for fluoxetine and 0.80+/-0.06 microM and 1.65+/-0.08 for norfluoxetine at +40 mV, respectively. In inside-out patches, norfluoxetine applied to the cytoplasmic surface inhibited Kv3.1 with an IC(50) value of 0.19+/-0.01 microM. The inhibition of Kv3.1 currents by both drugs was characterized by an acceleration in the apparent rate of current decay, without modification of the activation time course and with relatively fewer effects on peak amplitude. The degree of inhibition of Kv3.1 by norfluoxetine was voltage-dependent. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.31+/-0.05. The association (k(+1)) and dissociation (k(-1)) rate constants for norfluoxetine-induced inhibition of Kv3.1 were 21.70+/-3.39 microM(-1) s(-1) and 14.68+/-3.94 s(-1), respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 0.68 microM. Norfluoxetine did not affect the ion selectivity of Kv3.1. The reversal potential under control conditions was about -85 mV and was not affected by norfluoxetine. Norfluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of norfluoxetine, were superimposed. The voltage dependence of steady-state inactivation was not changed by the drug. Norfluoxetine produced use-dependent inhibition of Kv3.1 at a frequency of 1 Hz and slowed the recovery from inactivation. It is concluded that at clinically relevant concentrations, both fluoxetine and its major metabolite norfluoxetine inhibit Kv3.1, and that norfluoxetine directly inhibits Kv3.1 as an open channel blocker.


Assuntos
Fluoxetina/farmacologia , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Algoritmos , Animais , Encéfalo/citologia , Clonagem Molecular , Fluoxetina/análogos & derivados , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Canais de Potássio Shaw
9.
Neuropharmacology ; 41(3): 384-91, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11522330

RESUMO

To assess a possible role for phospholipase D (PLD) in PC12 cell signal transduction and differentiation, we have investigated the expression of PLD in PC12 cells and found that the differentiation factor, nerve growth factor (NGF) increased PLD1 protein expression and phorbol 12-myristate 13 acetate (PMA)-induced PLD activity. During neuronal differentiation, this effect showed correlation to the protein expression levels of classical protein kinase C (PKC) isozymes, PKC-alpha and -beta II, but there was no significant increase in the protein level of RhoA, another regulatory factor for PLD activation. Interestingly, PLD1 was associated with PKC-alpha or beta II, and its association gradually increased as NGF-induced neuronal differentiation progressed. PKC inhibitor, Ro-31-8220, caused a significant inhibition of neurite outgrowth and PLD activity. Furthermore, PLD1 was constitutively associated with the Shc adaptor molecule, the overexpression of which is known to induce PLD activity and to induce neurite outgrowth. Taken together, the data in this study suggests that PLD1 is closely implicated in neuronal differentiation of PC12 cells.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Neurônios/enzimologia , Fosfolipase D/biossíntese , Sequência de Aminoácidos , Animais , Western Blotting , Diferenciação Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Dados de Sequência Molecular , Células PC12 , Fosfolipase D/genética , Proteína Quinase C/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
10.
J Med Chem ; 41(18): 3435-41, 1998 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-9719596

RESUMO

A series of 2-amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (4-10) and 2-amino-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (1) were synthesized as potential prodrugs of penciclovir and evaluated for their oral penciclovir bioavailability in mice and rats. Treatment of 2-(2-benzyloxyethyl)propane-1,3-diol (11) with 1,1'-carbonyldiimidazole in THF followed by hydrogenolytic removal of the benzyl group of the resulting cyclic carbonate 12 gave 5-(2-hydroxyethyl)-1,3-dioxan-2-one (13). Mesylation of the alcohol 13 and then a coupling reaction of the resulting mesylate 14 with 2-amino-6-chloropurine using anhydrous Cs2CO3 in DMF afforded 2-amino-6-chloro-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (16) after purification by flash column chromatography on silica gel using EtOAc/MeCN/Et3N as eluent. Hydrogenation of the 6-chloro cyclic carbonate 16 followed by a ring-opening reaction of the 6-deoxy cyclic carbonate 1 in a mixture of an appropriate alcohol and CHCl3 using activated SiO2 as a Lewis acid afforded the corresponding alkyl monocarbonate derivatives 3-10 in fair to good yields. Of the prodrugs tested in mice, the isopropyl monocarbonate 6 achieved the highest mean urinary recovery of penciclovir (53%), followed in order by the propyl monocarbonate 5 (51%), the isopentyl monocarbonate 10 (51%), the ethyl monocarbonate 4 (50%), and famciclovir (48%). In rats, the methyl monocarbonate 3, 4, 6, the n-butyl monocarbonate 7, and 10 (39-41%) showed levels of mean urinary recovery of penciclovir similar to that from famciclovir (40%). The alkyl monocarbonates 4-10 were found to be quite stable in the aqueous buffer solutions, and among them, 6 was the most stable with the half-lives (t1/2) of 88, >200, 61, and 26 days at pH 1.2, 6.0, 7.4, and 8.0, respectively. In addition, 6 was highly soluble in H2O (138.8 mg/mL, 20 degrees C).


Assuntos
Aciclovir/análogos & derivados , Antivirais , Pró-Fármacos , Purinas , Aciclovir/química , Aciclovir/farmacocinética , Aciclovir/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Linhagem Celular Transformada , Chlorocebus aethiops , Estabilidade de Medicamentos , Guanina , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Masculino , Camundongos , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Purinas/síntese química , Purinas/química , Purinas/farmacocinética , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Solubilidade , Células Vero , Replicação Viral/efeitos dos fármacos
11.
Biochem Pharmacol ; 61(8): 1029-32, 2001 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11286994

RESUMO

The effects of H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), a potent and selective inhibitor of protein kinase A (PKA), were examined on Kv1.3 channels stably expressed in Chinese hamster ovary (CHO) cells using the patch clamp technique. In whole-cell recordings, H-89 decreased Kv1.3 currents and accelerated the decay rate of current inactivation in a concentration-dependent manner with an IC(50) value of 1.70 microM. These effects were completely reversible after washout. Intracellular infusion with PKA inhibitors, adenosine 3', 5'-cyclic phosphorothioate-Rp (Rp-cAMPS) or protein kinase A inhibitor 5-24 (PKI 5-24) had no effect on Kv1.3 currents and did not prevent the inhibitory action of H-89 on the current. H-89 applied to the cytoplasmic surface also inhibited Kv1.3 currents in excised inside-out patches. These findings suggest that H-89 inhibits Kv1.3 currents independently of PKA.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoquinolinas/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Sulfonamidas , Animais , Células CHO , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Canal de Potássio Kv1.3 , Canais de Potássio/metabolismo
12.
Biochem Pharmacol ; 62(5): 527-35, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11585049

RESUMO

The interaction of (-)-epigallocatechin-3-gallate (EGCG), the main component of green tea (Camellia sinensis), with rat brain Kv1.5 channels (rKv1.5) stably expressed in Chinese hamster ovary (CHO) cells was investigated using the whole-cell patch-clamp technique. EGCG inhibited rKv1.5 currents at +50 mV in a concentration-dependent manner, with an IC50 of 101.2+/-6.2 microM. Pretreatment with protein tyrosine kinase (PTK) inhibitors (10 microM genistein, 100 microM AG1296), a tyrosine phosphatase inhibitor (500 microM sodium orthovanadate), or a protein kinase C (PKC) inhibitor (10 microM chelerythrine) did not block the inhibitory effect of EGCG on rKv1.5. The inhibition of rKv1.5 by EGCG displayed voltage-independence over the full activation voltage range positive to +10 mV. EGCG had no effect on the midpoint potential or the slope factor for steady-state activation and inactivation. EGCG did not affect the ion selectivity of rKv1.5. The activation (at +50 mV) kinetics was significantly slowed by EGCG. During repolarization (at -40 mV), EGCG also slowed the deactivation of the tail currents, resulting in a crossover phenomenon. Reversal of inhibition was detected by the application of repetitive depolarizing pulses and of identical double pulses, especially during the early part of the activating pulse, in the presence of EGCG. EGCG-induced inhibition of rKv1.5 showed identical affinity between EGCG and the multiple closed states of rKv1.5. These results suggest that EGCG interacts directly with rKv1.5 channels. Furthermore, by analyzing the kinetics of the interaction between EGCG and rKv1.5, we conclude that the inhibition of rKv1.5 channels by EGCG includes at least two effects: EGCG preferentially binds to the channel in the closed state, and blocks the channel by pore occlusion while depolarization is maintained.


Assuntos
Catequina/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Chá/química , Animais , Anticarcinógenos/farmacologia , Células CHO , Catequina/análogos & derivados , Cricetinae , Relação Dose-Resposta a Droga , Transporte de Íons/efeitos dos fármacos , Cinética , Canal de Potássio Kv1.5 , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Ratos
13.
Histol Histopathol ; 19(4): 1015-9, 2004 10.
Artigo em Inglês | MEDLINE | ID: mdl-15375744

RESUMO

The expression and potential role of phospholipase D1 (PLD1) were studied in the cerebral cortex of rats after freeze injury. Histopathologically, cryoinjury, by exposing cerebral cortex to a prechilled rod for 1 minute, produced consistent pathological lesions, specifically neuronal death, infiltration of macrophages into the center of the cryoinjury, and reactive astrogliosis at the periphery, which caused the lesion site to become encased. Western blot analysis showed that PLD1 expression in the ipsilateral cerebral cortex increased significantly during days 1 to 3 after cryoinjury and declined slightly at post-injury day 7. PLD1 immunoreactivity was very low in the brains of sham-operated control adults. After cryoinjury, there was substantial PLD1 immunostaining of numerous inflammatory cells in the ipsilateral cortex, which were identical to ED1-positive macrophages. In addition, PLD1 immunoreactivity was increased in some neurons and astrocytes at the periphery of the cryoinjury at post-injury days 3 and 7. These findings suggest that cryoinjury by means of prechilled rods induced consistent histopathological changes in the cerebral cortex. In addition, expression of a cell activation signal, PLD1, was upregulated in macrophages and astrocytes in the ipsilateral cerebral cortex after cryoinjury.


Assuntos
Córtex Cerebral/enzimologia , Córtex Cerebral/lesões , Fosfolipase D/metabolismo , Animais , Western Blotting , Córtex Cerebral/patologia , Congelamento , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
14.
Mol Cells ; 11(3): 369-78, 2001 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-11459228

RESUMO

The regulatory mechanism through which the phospholipase D (PLD) isoforms PLD1 and PLD2 are activated is poorly understood. We investigated the possibility that the PLD isozymes are differentially regulated in response to pharmacologic stimulants in cells. In this report, we demonstrate for the first time that H2O2 and EGF differentially induce tyrosine phosphorylation of the PLD isozymes in A431 cells, which express both PLD1 and PLD2. H2O2 induced tyrosine phosphorylation of PLD1 and PLD2, whereas EGF only caused the tyrosine phosphorylation of PLD2. Both agents also induced phosphorylation of the EGF receptor. Interestingly, the PLD isozymes were associated with the EGF receptor and PKC-alpha in a ligand independent manner. Activation of PLD by H2O2 and EGF nearly correlated with tyrosine phosphorylation of the protein in PLD1 immune complexes. Activation of PLD by both agents was inhibited by the PKC inhibitor, Ro 31-8220, and by the down-regulation of PKC. Pretreatment of the cells with the tyrosine kinase inhibitor tyrphostin AG1478 resulted in inhibition of the H2O2 and EGF-induced tyrosine phosphorylation and PLD activation. These results indicate that H2O2 and EGF induce differential tyrosine phosphorylation of PLD isozymes. Also, the activation of PLD by these agonists involves tyrosine phosphorylation and PKC activation.


Assuntos
Carcinoma de Células Escamosas , Fator de Crescimento Epidérmico/farmacologia , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Fosfolipase D/metabolismo , Tirosina/metabolismo , Catalase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Isoenzimas/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Células Tumorais Cultivadas
15.
Mol Cells ; 11(3): 386-91, 2001 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-11459230

RESUMO

To clarify the involvement of phospholipase D (PLD) in the mechanism underlying genetically-induced hypertension, we investigated the activity and expression levels of PLD in tissues taken from spontaneously hypertensive rats (SHR), and their normotensive controls, Wistar-Kyoto rats (WKY). The ADP-ribosylation factor 3 (ARF3)-dependent PLD activity and protein levels of PLD1 from SHR increased significantly in the brain and liver, but not in the heart and kidney, compared to those of WKY. The activity and expression of PLD were the same between the homogenated whole kidneys of the two strains; however, there were topographical differences in the expression and activity of PLD between the kidneys of the two strains. The activity and expression level of PLD gradually increased from the cortex to the inner medulla of WKY. The enzyme activity, and amount of PLD in the inner stripe of the outer medulla and in the inner medulla, was significantly lower in SHR than in WKY. Taken together, these results suggest that the distinctly distributed patterns of PLD in the kidney may be associated with differential signal transduction pathways that are involved in hypertension in conjunction with an increase of PLD activity in the brain and liver.


Assuntos
Regulação Enzimológica da Expressão Gênica , Hipertensão/metabolismo , Fosfolipase D/genética , Animais , Encéfalo/enzimologia , Hipertensão/fisiopatologia , Rim/enzimologia , Fígado/enzimologia , Masculino , Miocárdio/enzimologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais/fisiologia
16.
Brain Res ; 905(1-2): 240-4, 2001 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-11423101

RESUMO

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the expression and cellular localization of the PLD isozyme PLD1 in the rodent retina. Western blot analysis showed the presence of PLD1 at the protein level in the rat, mouse and guinea pig retinas. PLD1 immunoreactivity was localized in all Müller cells. Thus, PLD1 protein appears to be important in the functions of these cells in the rodent retina.


Assuntos
Neuroglia/enzimologia , Fosfolipase D/metabolismo , Retina/enzimologia , Roedores/metabolismo , Animais , Expressão Gênica/fisiologia , Glutamato-Amônia Ligase/metabolismo , Cobaias , Imuno-Histoquímica , Camundongos , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Ratos , Retina/citologia , Roedores/anatomia & histologia , Visão Ocular/fisiologia
17.
Pancreas ; 22(1): 47-52, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11138970

RESUMO

Phospholipase C (PLC) isozymes are believed to play a role in regulating pancreatic exocrine and endocrine secretion. In an attempt to investigate the role of PLC, we examined the distribution patterns of PLC isozymes in the normal rat pancreas by Western blot analysis and immunohistochemistry. Western blot analysis was performed on pancreatic acinar tissues and the islet of Langerhans, which were separated from each other. PLC-beta isozymes (beta1, beta2, beta3, and beta4), delta1, and delta2 were detected in both acinar and islet cells, whereas PLC-gamma1 and gamma2 were observed only in acinar tissues. On immunohistochemistry, the immunoreactivities of PLC isozymes except for PLC-gamma1 were observed as follows: PLC-beta1, in both the exocrine and endocrine tissues; PLC-beta2, mainly in the periphery of the islet and acinar cells; PLC-beta3, in the periphery of the islet and in some ductal epithelium; PLC-beta4, through the islet of Langerhans and ductal epithelium; PLC-gamma1, not detected in pancreatic tissue; PLC-gamma2, mainly in acinar cells; PLC-delta1 and delta2, in the islet and in ductal epithelium. These results suggest that the intrapancreatic site-specific existence of PLC isozymes may modulate pancreatic exocrine and endocrine functions through a PLC-mediated signal transduction.


Assuntos
Pâncreas/enzimologia , Fosfolipases Tipo C/metabolismo , Animais , Western Blotting , Técnicas Imunoenzimáticas , Ilhotas Pancreáticas/enzimologia , Isoenzimas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
18.
Eur J Pharmacol ; 413(2-3): 281-5, 2001 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-11226404

RESUMO

To understand the underlying mechanism of ethanol in tonic contraction, the effect of ethanol on phospholipase A(2) and phospholipase C activities and the effects of phospholipase inhibitors on ethanol-induced contraction of cat gastric smooth muscle were tested. Circular muscle strips (2.0 x 0.2 cm) obtained from the fundus of cat stomach were used to measure isometric contraction. Ethanol elicited tonic contraction and activated phospholipase A(2) activity in a dose-dependent manner. Phospholipase A(2) inhibitors, manoalide (0.1--10 microM) and oleyloxyethyl phosphorylcholine (1--10 microM), significantly inhibited ethanol-induced contraction. Furthermore, 342 mM ethanol-induced contraction was significantly inhibited by cyclooxygenase inhibitors, ibuprofen (10--100 microM) and indomethacin (10--100 microM), but not by lipoxygenase inhibitors. On the other hand, phospholipase C inhibitors had no effect on ethanol-induced contraction, indicating that phospholipase C is not involved in ethanol-induced contraction. It is suggested from the above results that ethanol-induced contraction in cat gastric smooth muscle is, in part, mediated by phospholipase A(2) and cyclooxygenase pathways.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Fundo Gástrico/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Fosfolipases A/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Animais , Gatos , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Feminino , Fundo Gástrico/metabolismo , Ibuprofeno/farmacologia , Indometacina/farmacologia , Fosfatos de Inositol/metabolismo , Masculino , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Fosfolipases A/metabolismo , Fosforilcolina/farmacologia , Terpenos/farmacologia
19.
Pancreas ; 23(1): 102-8, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11451139

RESUMO

The effect of somatostatin on cholecystokinin-induced amylase release was investigated in isolated rat pancreatic acini. Acini were isolated by enzymatic digestion and incubated in a HEPES buffered Ringer's solution with testing reagents for 30 minutes at 37 degrees C. The activity of released amylase, cAMP, and inositol phosphate formation were measured. Intracellular calcium concentration ([Ca2+]i) was also checked. Somatostatin 14 and octreotide, a somatostatin analog, inhibited CCK-stimulated amylase release in a concentration-dependent manner. The inhibitory effect of octreotide on CCK-induced amylase release was not shown when the acini were treated with 8-Br-cAMP, irrespective of the presence of IBMX. Forskolin potentiated CCK-induced amylase release and this effect was blocked by octreotide treatment; although CCK-8 (3 x 10(-11) M) failed to stimulate cAMP formation, octreotide significantly inhibited basal cAMP formation in the acini. The increase of [Ca2+]i in response to CCK was inhibited by octreotide. However, CCK-induced inositol phosphate formation was not changed by 10(-9) M octreotide. Octreotide had no effect on CCK-stimulated tyrosine phosphorylation, and tyrosine phosphatase inhibitors (NaF and Na2WO4) did not influence the effect of octreotide on CCK-induced amylase release. From these results, we conclude that octreotide inhibits CCK-induced amylase release by inhibiting basal cAMP formation and decreasing the [Ca2+]i stimulated by CCK.


Assuntos
Amilases/metabolismo , Pâncreas/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sincalida/farmacologia , Somatostatina/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sistema Livre de Células , Colforsina/farmacologia , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fosfatos de Inositol/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Octreotida/farmacologia , Pâncreas/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/fisiologia , Ratos , Taxa Secretória/efeitos dos fármacos , Fluoreto de Sódio/farmacologia , Compostos de Tungstênio/farmacologia
20.
Neurosci Lett ; 316(2): 95-8, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11742724

RESUMO

Phospholipase D1 (PLD1) expression was studied in the central nervous system (CNS) under the condition of induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. After inducing EAE, the expression of PLD1 was analyzed by Western blot and immunohistochemistry. Western blot analysis showed that expression of the isozymes PLD1 significantly increased in the spinal cord at the peak stage of EAE, and declined thereafter. Immunohistochemistry showed that PLD1-positive cells increased in number in EAE lesions, which consisted mainly of ED1-positive macrophages and glial fibrillary acidic protein-positive astrocytes. In contrast, PLD1 was only weakly expressed in some spinal cord astrocytes in control rats. These results suggest that PLD1 is increased in autoimmune CNS inflammation, and possibly involved in the activation of macrophages and astrocytes in EAE lesions.


Assuntos
Astrócitos/enzimologia , Encefalomielite Autoimune Experimental/enzimologia , Gliose/enzimologia , Esclerose Múltipla/enzimologia , Fosfolipase D/metabolismo , Medula Espinal/enzimologia , Regulação para Cima/imunologia , Animais , Astrócitos/imunologia , Astrócitos/patologia , Western Blotting , Quimiotaxia de Leucócito/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/imunologia , Imuno-Histoquímica , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/patologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais/imunologia , Medula Espinal/imunologia , Medula Espinal/patologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/patologia
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