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1.
Toxicol In Vitro ; 21(7): 1258-61, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17553660

RESUMO

The aims of this work were to compare the effects of methyl mercury chloride and thimerosal on neurite/process outgrowth and microtubule proteins in differentiating mouse N2a neuroblastoma and rat C6 glioma cells. Exposure for 4h to sublethal concentrations of both compounds inhibited neurite outgrowth to a similar extent in both cells lines compared to controls. In the case of N2a cells, this inhibitory effect by both compounds was associated with a fall in the reactivity of western blots of cell extracts with monoclonal antibody T1A2, which recognises C-terminally tyrosinated alpha-tubulin. By contrast, reactivity with monoclonal antibody B512 (which recognises total alpha-tubulin) was unaffected at the same time point. These findings suggest that decreased tubulin tyrosination represents a neuron-specific early marker of mercury toxicity associated with impaired neurite outgrowth.


Assuntos
Compostos de Metilmercúrio/toxicidade , Timerosal/toxicidade , Tubulina (Proteína)/efeitos dos fármacos , Tirosina/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Biomarcadores , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Glioma/metabolismo , Camundongos , Proteínas dos Microtúbulos/efeitos dos fármacos , Proteínas dos Microtúbulos/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuroblastoma/metabolismo , Ratos , Tubulina (Proteína)/metabolismo
2.
FEBS Lett ; 464(1-2): 48-52, 1999 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-10611481

RESUMO

A selective cyclic nucleotide PDE5 inhibitor corrected the defective mucin secretion response to the beta-agonist isoproterenol in submandibular acinar cells inhibited by antibody directed against the cystic fibrosis transmembrane conductance regulator. The PDE5 inhibitor was as effective as cpt-cyclic AMP or a selective PDE4 inhibitor. However, the PDE5 inhibitor had no effect on basal or isoproterenol-stimulated cyclic AMP levels and did not stimulate mucin secretion. The results showing, for the first time, correction of the CFTR mucin secretion defect by a PDE5 inhibitor, which may involve cyclic GMP, will have a major impact in development of a rational drug treatment for cystic fibrosis.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Mucinas/metabolismo , Glândula Submandibular/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Anticorpos/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Humanos , Isoproterenol/farmacologia , Mucinas/efeitos dos fármacos , Purinonas/farmacologia , Piridazinas/farmacologia , Piridinas/farmacologia , Ratos , Rolipram/farmacologia , Glândula Submandibular/efeitos dos fármacos
3.
Br J Pharmacol ; 125(4): 697-704, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9831904

RESUMO

1. The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated beta-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited beta-adrenergic response and whether excessive cyclic AMP rise is required. 2. CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured. 3. The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion. 4. DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion. 5. CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism. 6. DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not. 7. Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of beta-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis.


Assuntos
AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/farmacologia , Mucinas/metabolismo , Antagonistas de Receptores Purinérgicos P1 , Receptores Adrenérgicos beta/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/química , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Anticorpos/farmacologia , Cromatografia em Gel , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Relação Dose-Resposta a Droga , Eletroforese em Acetato de Celulose , Mucinas/efeitos dos fármacos , Fosforilação , Testes de Precipitina , Ratos , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/fisiologia , Teobromina/análogos & derivados , Teobromina/química , Teobromina/farmacologia , Teofilina/análogos & derivados , Teofilina/química , Teofilina/farmacologia
4.
Biochem J ; 324 ( Pt 2): 645-51, 1997 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9182729

RESUMO

It is recognized in many cellular systems that the receptor/G-protein activation of phospholipase C and Ins(1,4,5)P3 production is the transduction pathway regulating the release of Ca2+ from internal stores. Ca2+ signals can now be monitored at the level of single cells but the biochemical detection of Ins(1,4,5)P3 cannot match this resolution. It is often difficult or impossible to directly attribute responses evoked in single cells by putative phospholipase C-coupled agonists to changes in Ins(1,4,5)P3 levels. U73122 is an aminosteroid that is reported to act as a specific inhibitor of phospholipase C and it has become an important tool in establishing the link between phospholipase C activation and cellular Ca2+ signalling. In the present study we use both patch-clamp electrophysiology and the imaging of fluorescent Ca2+ indicators to investigate the effect of U73122 in mouse pancreatic acinar cells. The study reveals that U73122 has effects other than the inhibition of phospholipase C. U73122 can directly activate ion channels. It can itself promote the release of Ca2+ from intracellular stores in permeabilized cells and in intact cells it triggers a release of Ca2+ that is initiated specifically at the secretory pole of these morphologically and functionally polarized cells. We also present evidence that U73122 can potentiate the response to Ins(1,4,5)P3; this is seen both in permeabilized cells and in patch-clamp protocols in which cells are internally dialysed with submaximal concentrations of Ins(1,4,5)P3. The effects of U73122 are therefore multiple and not specific for the inhibition of phospholipase C. Importantly, all the effects described influence Ca2+ signalling yet in many experimental protocols some of these effects can go unnoticed and might in error be attributed simply to the inhibition of Ins(1,4,5)P3 production.


Assuntos
Cálcio/metabolismo , Estrenos/farmacologia , Inositol 1,4,5-Trifosfato/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Sistemas do Segundo Mensageiro/fisiologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/fisiologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Digitonina/farmacologia , Líquido Intracelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Técnicas de Patch-Clamp , Fosfatidilinositol Diacilglicerol-Liase
5.
Biochem J ; 293 ( Pt 3): 691-5, 1993 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-8102525

RESUMO

Increases in cytoplasmic free Ca2+ concentration in rat submandibular acini were observed in response to isoprenaline (10 microM), noradrenaline (10 microM) and carbamoylcholine (10 microM). Noradrenaline and carbamoylcholine responses were decreased to 27% and 33% respectively in the absence of extracellular Ca2+, suggesting a major requirement for Ca2+ entry. beta-Adrenergic stimulation elicited a small (35-40 nM) free Ca2+ rise, approx. 75% of which was mobilized from an intracellular store. Results suggest that this Ca2+ rise is a key event in the physiological triggering of mucin secretion by exocytosis.


Assuntos
Cálcio/metabolismo , Receptores Adrenérgicos beta/fisiologia , Glândula Submandibular/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , AMP Cíclico/metabolismo , Homeostase , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Mucinas/metabolismo , Ratos , Ratos Wistar , Glândula Submandibular/efeitos dos fármacos
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