Functional IP3- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108-15 (rodent neuroblastoma x glioma hybrid) cells

Abstract

1. 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. 2. Initial demonstration of the presence of IP3 receptors and ryanodine receptors in the NG108-15 varicosities was obtained using immunocytochemistry. 3. Treatment of NG108-15 cells with bradykinin (0.1 μM), 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. 4. 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 μM). 5. Bradykinin-induced changes in [Ca2+]i were abolished following depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 μM) and were reduced (72%) by prior emptying of the ryanodine-sensitive Ca2+ store with caffeine (10 mM). 6. The same results were obtained when the varicosities of the NG108-15 cells had formed synaptic junctions with co-cultured rat hindlimb myotubes. 7. 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.