RESUMO
Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.
Assuntos
Melanotrofos/metabolismo , Proteínas rab3 de Ligação ao GTP/fisiologia , Proteína rab3A de Ligação ao GTP/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Capacitância Elétrica , Exocitose/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Injeções , Melanotrofos/efeitos dos fármacos , Melanotrofos/fisiologia , Microscopia Confocal , Oligonucleotídeos Antissenso/farmacologia , Técnicas de Patch-Clamp , Hipófise/metabolismo , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/metabolismo , Distribuição Tecidual , Proteínas rab3 de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/administração & dosagem , Proteína rab3A de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/farmacologiaRESUMO
We report the sequence, structure and distribution of a novel transient receptor potential (trp) homologue from Xenopus, Xtrp, determined by screening an oocyte cDNA library. On the basis of sequence similarity and predicted structure, Xtrp appears to be a homologue of mammalian trp1 proteins. Two polyclonal antibodies raised against distinct regions of the Xtrp sequence revealed Xtrp expression in various Xenopus tissues, and the localization of Xtrp at the plasma membrane of Xenopus oocytes and HeLa cells. Since capacitative calcium entry into Xenopus oocytes has been shown previously to be substantially inhibited by trp1 antisense oligonucleotides [Tomita, Kaneko, Funayama, Kondo, Satoh and Akaike (1998) Neurosci. Lett. 248, 195-198] we suggest that Xtrp may underlie capacitative calcium entry in Xenopus tissues.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas de Drosophila , Proteínas de Membrana/genética , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Anticorpos , Sequência de Bases , Proteínas de Ligação a Calmodulina/análise , Proteínas de Ligação a Calmodulina/química , Membrana Celular/química , Clonagem Molecular , Biblioteca Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas de Membrana/análise , Proteínas de Membrana/química , Dados de Sequência Molecular , Peso Molecular , Oócitos/citologia , Oócitos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Canais de Potencial de Receptor TransitórioRESUMO
Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.
