Comparison of two techniques in the evaluation of calcium transients elicited by metabotropic purinergic receptors in glial satellite cells (2013)
- Autor USP: CASSOLA, ANTONIO CARLOS - ICB
- Unidade: ICB
- Subjects: FISIOLOGIA; CALCIO
- Language: Português
- Abstract: Introdução Dorsal root ganglia (DRG) contains pseudo-unipolar neurons of sensory input. Neuron somata is enveloped by satellite glial cells (SGC) whose functions is unclear. A recent report has shown a paracrine communication between neurons and SGC mediated by ionotropic purinergic receptors (PNAS 104:9864, 2007). In previous studies we demonstrated that SGC from rats express several subtypes of P2Y, metabotropic purinergic receptor. The study was carried on observing intracelular calcium transients elicited by especific agonists and antagonists of P2Y receptors. Fluorescence signals from Fluo-4 (Molecular Probes), a calcium indicator, were detected by a confocal laser scanning microscope (Zeiss, LSM 510). Despite the consistency of many measurements showing a rapid rise of the intracellular free-Ca2+ concentration ([Ca2+]i) and a slow decay, upon fast application of agonists, some concern about the proper follow-up of the kinetics remain. The concern arise from the t: a) The limited time resolution of the confocal scanning (0.33 Hz) and from the use of Fluo-4, whose excitation-emission properties precludes the calculation of fluorescence raios. Objetivos Here we show intracellular free-Ca2+ measurements with a wide-field fluorescence microscope with a better time resolution (2Hz). Besides the transients were observed with Fura-2 (Molecular Probes), a ratiometric indicator.Métodos DRG from newborn (3 days) Wistar rats were treatead with collagenase and mechanically dissociated. The cells were cultivated on poli-L-lysine-treated glass cover slips. The culture was kept in DMEM with 5% fetal bovine serum, at 37°C and under 5% CO2 atmosphere. Satellite cells still attached to neurons were observed. Changes in [Ca2+]i were evaluted in a fluorescence microscope with UV ilumination (Leica, AF6000), using Fura-2 as a calcium indicator. Fluorescence was excited at 340 nm (F340) and 387 nm (F387) and emitted fluorescence observed above 510 nm. The fluorescence ratio F340/F387 (R) was used to describe relative changes in the [Ca2+]i. Time series were recorded in control conditions and during the exposure to the agonist. Resultados ATP 0.1 mM promoted transitory increases in the [Ca2+]i in SGC (n=53). The medium value of R at the peck is 1.399 with a standart deviation of ± 0.402. Comparing the kinetics of estimates with Fluo-4 and Fura-2, the overlap is such that artifacts in the previous measurements are excluded. Conclusão Coincidence in the observed free-Ca2+ transients by two techniques strengthen previous evidences for the occurrence of several types of purinergic receptor in satellite glial cells.
- Título do periódico: Resumos
- Conference titles: Reunião Anual da Federação de Sociedades de Biologia Experimental (FeSBE)
ABNTBATISTA, D. R.; CASSOLA, Antonio Carlos. Comparison of two techniques in the evaluation of calcium transients elicited by metabotropic purinergic receptors in glial satellite cells. Anais.. Caxambu: FeSBE, 2013.
APABatista, D. R., & Cassola, A. C. (2013). Comparison of two techniques in the evaluation of calcium transients elicited by metabotropic purinergic receptors in glial satellite cells. In Resumos. Caxambu: FeSBE.
NLMBatista DR, Cassola AC. Comparison of two techniques in the evaluation of calcium transients elicited by metabotropic purinergic receptors in glial satellite cells. Resumos. 2013 ;
VancouverBatista DR, Cassola AC. Comparison of two techniques in the evaluation of calcium transients elicited by metabotropic purinergic receptors in glial satellite cells. Resumos. 2013 ;
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