A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca2+ signaling
Por:
Cifuentes F., González C.E., Fiordelisio T., Guerrero G., Lai F.A., Hernández-Cruz A.
Publicada:
1 ene 2001
Resumen:
The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca2+ release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC50 of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca2+ release; (2) diminished voltage-gated Ca2+ influx, due to a positive shift in the activation of voltage gated Ca2+ currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR2 antibodies, but evidence of caffeine-induced Ca2+ release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca2+ signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca2+ release, and the Ca2+ release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca2+ homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis. © 2001 Elsevier Science Inc.
Filiaciones:
Cifuentes F.:
Departamento de Biofísica, Inst. Fisiol. Cel., Univ. Nac. A., México City, Mexico
González C.E.:
Departamento de Biofísica, Inst. Fisiol. Cel., Univ. Nac. A., México City, Mexico
Fiordelisio T.:
Departamento de Biofísica, Inst. Fisiol. Cel., Univ. Nac. A., México City, Mexico
Guerrero G.:
Departamento de Biofísica, Inst. Fisiol. Cel., Univ. Nac. A., México City, Mexico
Lai F.A.:
Department of Cardiology, Univ. Wales Coll. Med., Hlth. P., Cardiff, United Kingdom
Hernández-Cruz A.:
Departamento de Biofísica, Inst. Fisiol. Cel., Univ. Nac. A., México City, Mexico
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