RESUMO
The five different rat somatostatin receptor subtypes (SSTR1-SSTR5) were coexpressed with a subunit of G-protein gated inwardly rectifying potassium channel (GIRK1) in Xenopus oocytes. SSTR2-SSTR5, but not SSTR1 coupled efficiently to the activation of GIRK currents when stimulated by SST14 or SST28. A comparison of the dose-response curves and of the maximum currents obtained indicates that SSTR2 couples most efficiently to this effector, supporting the notion that SSTR2 is involved in activation of potassium conductances by SST in vivo.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Receptores de Somatostatina/fisiologia , Somatostatina/farmacologia , Animais , Compostos de Bário/farmacologia , Cloretos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Oócitos , Técnicas de Patch-Clamp , Canais de Potássio/genética , Ratos , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/genética , Somatostatina-28 , Xenopus laevisRESUMO
BACKGROUND AND PURPOSE: Episodic phases of continuous poor-quality oocytes obtained from South American Clawed Frogs (Xenopus laevis) often are observed. In publications dealing with the surgical technique of oocyte removal, the frogs' robust constitution and resistance against infections provided by magainins are pointed out. For this reason, clean rather than sterile conditions for the surgical procedure are mostly recommended. However, in most instances, antibiotics are added to the buffer medium when in vitro experiments are performed using oocytes. METHODS: After a long phase of poor oocyte quality at our facility, involving oocytes that had been obtained by use of a "clean" surgical procedure, we subsequently cultured oocytes in a buffer medium containing the three antibiotics: penicillin G, gentamicin, and streptomycin. RESULTS: During DNA injection experiments, the oocytes developed black spots on their surface by postoperative day two. Pure culture of the gram-negative non-fermentative rod Pseudomonas fluorescens was obtained from the impaired oocytes; the isolate was resistant to the three antibiotics. By contrast, after aseptic surgical removal and culture of oocytes in buffer medium containing the antibiotics tetracycline and gentamicin, perfect oocytes without bacterial contamination were obtained. CONCLUSION: Whenever impaired oocyte quality is observed, microbial contamination should be considered as a possible cause.
Assuntos
Antissepsia/métodos , Assepsia/métodos , Oócitos/citologia , Oócitos/microbiologia , Ovariectomia/métodos , Pseudomonas fluorescens/isolamento & purificação , Coleta de Tecidos e Órgãos/métodos , Animais , Células Cultivadas , Técnicas Citológicas , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos , Cirurgia Geral/métodos , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Ovariectomia/normas , Penicilina G/farmacologia , Pseudomonas fluorescens/efeitos dos fármacos , Pele/microbiologia , Estreptomicina/farmacologia , Tetraciclina/farmacologia , Coleta de Tecidos e Órgãos/normas , Xenopus laevisRESUMO
By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile alpha motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.