Origin sites of calcium release and calcium oscillations in frog sympathetic neurons.
In many neurons, Ca(2+) signaling depends on efflux of Ca(2+) from intracellular stores into the cytoplasm via caffeine-sensitive ryanodine receptors (RyRs) of the endoplasmic reticulum. We have used high-speed confocal microscopy to image depolarization- and caffeine-evoked increases in cytoplasmic Ca(2+) levels in individual cultured frog sympathetic neurons. Although caffeine-evoked Ca(2+) wave fronts propagated throughout the cell, in most cells the initial Ca(2+) release was from one or more discrete sites that were several micrometers wide and located at the cell edge, even in Ca(2+)-free external solution. During cell-wide cytoplasmic [Ca(2+)] oscillations triggered by continual caffeine application, the initial Ca(2+) release that began each Ca(2+) peak was from the same subcellular site or sites. The Ca(2+) wave fronts propagated with constant amplitude; the spread was mostly via calcium-induced calcium release. Propagation was faster around the cell periphery than radially inward. Local Ca(2+) levels within the cell body could increase or decrease independently of neighboring regions, suggesting independent action of spatially separate Ca(2+) stores. Confocal imaging of fluorescent analogs of ryanodine and thapsigargin, and of MitoTracker, showed potential structural correlates to the patterns of Ca(2+) release and propagation. High densities of RyRs were found in a ring around the cell periphery, mitochondria in a broader ring just inside the RyRs, and sarco-endoplasmic reticulum Ca(2+) ATPase pumps in hot spots at the cell edge. Discrete sites at the cell edge primed to release Ca(2+) from intracellular stores might preferentially convert Ca(2+) influx through a local area of plasma membrane into a cell-wide Ca(2+) increase.
McDonough
S I
SI
Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201, USA.
Cseresnyés
Z
Z
Schneider
M F
MF
D000814:Aniline Compounds; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005456:Fluorescent Dyes; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D018715:Microscopy, Video; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011894:Rana pipiens; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D013564:Sympathetic Nervous System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014741:Video Recording; D014966:Xanthenes
D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D014966:Xanthenes; D002110:Caffeine; D000252:Calcium-Transporting ATPases; D002118:Calcium