P. Ronde et al., Functional IP3- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108-15 (rodent neuroblastoma x glioma hybrid) cells, J PHYSL LON, 529(2), 2000, pp. 307-319
1. Presynaptic varicosities of the model neuronal cell line NG108-15, a cho
linergic neuroblastoma cell x glioma cell hybrid capable of innervating str
iated myotubes, were examined for the presence of inositol 1,4,5-trisphosph
ate (IP3)-sensitive and Ca2+-activated (ryanodine-sensitive) Ca2+ stores us
ing confocal microscopic imaging of Ca2+-sensitive fluorescent dye loaded i
nto the cells.
2. Initial demonstration of the presence of IP3 receptors and ryanodine rec
eptors in the NG108-15 varicosities was obtained using immunocytochemistry.
3. Treatment of NG108-15 cells with bradykinin (0.1 muM), whose receptor is
linked to IP3 generation, and separately, caffeine (10 mM), an activator o
f endoplasmic reticulum ryanodine receptors, resulted in substantial increa
ses in [Ca2+](i) in the varicosities.
4. K+-evoked changes in [Ca2+](i) in the varicosities were reduced (52%) af
ter emptying the ryanodine-sensitive Ca2+ store using caffeine (10 mM), but
were not affected by prior depletion of IP3-sensitive Ca2+ store using tha
psigargin (1 muM).
5. Bradykinin-induced changes in [Ca2+](i) were abolished following depleti
on of IP3-sensitive Ca2+ store using thapsigargin (1 muM) 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 cel
ls had formed synaptic junctions with co-cultured rat hindlimb myotubes.
7. Taken together, the results suggest that, in the varicosities, activatio
n of the IP3 pathway evoked the release of Ca2+ from the IP3-sensitive stor
e, which, in turn, secondarily induced the release of Ca2+ from the ryanodi
ne-sensitive store via Ca2+-induced Ca2+ release, and that depolarization-i
nduced Ca2+ entry evoked Ca2+-induced Ca2+ release only from the ryanodine-
sensitive store. Thus, functional internal Ca2+ stores are inherent compone
nts of presynaptic varicosities in this neural cell line.