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 Ca(2+) signaling.
The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) 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 IC(50) 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 Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) 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-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) 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 Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) 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.
Cifuentes
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Departamento de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, PO Box 70-253, D.F. 04510, México City, Mexico.
González
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CE
Fiordelisio
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T
Guerrero
G
G
Lai
F A
FA
Hernández-Cruz
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A
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000918:Antibody Specificity; D001896:Boron Compounds / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism*; D018942:Macrolides; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051381:Rats; D012433:Ryanodine / Q000031:analogs & derivatives* / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D017783:Superior Cervical Ganglion / Q000166:cytology
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000900:Anti-Bacterial Agents; D001896:Boron Compounds; D015220:Calcium Channels; D005456:Fluorescent Dyes; D018942:Macrolides; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D020126:Brefeldin A; D002110:Caffeine; D002118:Calcium