RESUMO
Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of < or =20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K(+) and 2 mM Ca(2+)) provoking intense exocytosis (51 +/- 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 +/- 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots ( approximately 0.7 microm) selectively labeled by 40- to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded approximately 20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions.
Assuntos
Glândulas Suprarrenais/metabolismo , Células Cromafins/metabolismo , Endocitose , Endossomos/metabolismo , Exocitose , Vesículas Transportadoras/metabolismo , Acetilcolina/farmacologia , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Azul de Bromofenol/química , Cálcio/metabolismo , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/enzimologia , Ciclodextrinas/química , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Corantes Fluorescentes/química , Homeostase , Fusão de Membrana , Camundongos , Nicotina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Potássio/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Compostos de Piridínio/química , Compostos de Amônio Quaternário/química , Coloração e Rotulagem/métodos , Fatores de Tempo , Vesículas Transportadoras/efeitos dos fármacosRESUMO
Lead (Pb2+) is a cytotoxic metal ion whose mechanism of action is not established. However, Pb2+ is known to interact with a wide variety of molecules involved in signal transduction. In this study the effect of Pb2+ on protein phosphorylation in bovine adrenal chromaffin cells and human SH SY5Y cells was examined. Cells were incubated with 32P(i) for 1 h in the presence of Pb2+ (1-10 microM) and the proteins were separated by two-dimensional PAGE. An increase in the phosphorylation of a number of proteins was observed in response to Pb2+, including three spots, MW 25 kDa, and pI's in the range 4.0-4.5. These proteins were immunoidentified as three isoforms of the heat-shock protein 27 kDa (Hsp27), and the identity of the most basic spot was confirmed by amino acid sequencing. Phosphorylation of p38MAPK was increased by Pb2+ and the effect of Pb2+ on Hsp27 phosphorylation was blocked by the p38MAPK inhibitor SB203580 (1 microM). The results were similar for bovine chromaffin cells and human SH SY5Y cells. This is the first report showing that Pb2+ can modulate the phosphorylation state of Hsp27 via activation of the p38MAPK pathway.
Assuntos
Chumbo/toxicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Autorradiografia , Bovinos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/enzimologia , Células Cromafins/metabolismo , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Fosfatos/metabolismo , Fosforilação , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Because of their lack of long-term viability, adrenal tissue transplants have shown limited success in alleviating the motor disturbances associated with experimental and pathologic striatal dopamine denervation. In this study, we examined how the graft placement site influences adrenal medulla transplant survival and its relation with the reduction of motor deficits in rats bearing unilateral 6-OHDA lesion. METHODS: One or 5 microL of fetal adrenal medullar tissue was grafted either inside the striatal parenchyma or into the lateral ventricle in contact with the dopamine-denervated striatum. Motor disturbances, as assessed by apomorphine-induced rotation, were correlated to the graft morphologic survival features. RESULTS: Apomorphine-induced rotation showed a marginal reduction of 11% in all groups independently of graft survival features or placement site. Intrastriatal transplants showed limited viability characterized by a substantial loss of graft initial volume as well as fewer and smaller chromaffin cells compared to ventricular grafts, which had a reduced loss of graft initial volume and more and larger chromaffin cells. CONCLUSIONS: Although the lateral ventricle may favor adrenal medulla transplant viability, their induced motor outcome is comparable to that induced by less viable intrastriatal grafts, suggesting that the implanted dopamine-producing cells may interact and influence striatal neurons better when placed in close proximity.