Functional IP3- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108-15 (rodent neuroblastoma x glioma hybrid) cells.
Presynaptic varicosities of the model neuronal cell line NG108-15, a cholinergic neuroblastoma cell x glioma cell hybrid capable of innervating striated myotubes, were examined for the presence of inositol 1,4,5-trisphosphate (IP3)-sensitive and Ca2+-activated (ryanodine-sensitive) Ca2+ stores using confocal microscopic imaging of Ca2+-sensitive fluorescent dye loaded into the cells. Initial demonstration of the presence of IP3 receptors and ryanodine receptors in the NG108-15 varicosities was obtained using immunocytochemistry. Treatment of NG108-15 cells with bradykinin (0.1 microM), whose receptor is linked to IP3 generation, and separately, caffeine (10 mM), an activator of endoplasmic reticulum ryanodine receptors, resulted in substantial increases in [Ca2+]i in the varicosities. K+-evoked changes in [Ca2+]i in the varicosities were reduced (52 %) after emptying the ryanodine-sensitive Ca2+ store using caffeine (10 mM), but were not affected by prior depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 microM). Bradykinin-induced changes in [Ca2+]i were abolished following depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 microM) and were reduced (72 %) by prior emptying of the ryanodine-sensitive Ca2+ store with caffeine (10 mM). The same results were obtained when the varicosities of the NG108-15 cells had formed synaptic junctions with co-cultured rat hindlimb myotubes. Taken together, the results suggest that, in the varicosities, activation of the IP3 pathway evoked the release of Ca2+ from the IP3-sensitive store, which, in turn, secondarily induced the release of Ca2+ from the ryanodine-sensitive store via Ca2+-induced Ca2+ release, and that depolarization-induced Ca2+ entry evoked Ca2+-induced Ca2+ release only from the ryanodine-sensitive store. Thus, functional internal Ca2+ stores are inherent components of presynaptic varicosities in this neural cell line.
Rondé
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Departments of Pharmacology and Physiology, and Neurobiology and Anatomy, Medical College of Pennsylvania Hahnemann University, Philadelphia, PA 19102, USA.
Dougherty
J J
JJ
Nichols
R A
RA
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D005910:Glioma; D006822:Hybrid Cells; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008564:Membrane Potentials; D008959:Models, Neurological; D018482:Muscle, Skeletal / Q000294:innervation / Q000378:metabolism; D009447:Neuroblastoma; D011188:Potassium / Q000378:metabolism; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D011188:Potassium; D001920:Bradykinin; D002118:Calcium