Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.

Fura-2 imaging microscopy was used to study [Ca2+]i in nerve growth factor-differentiated PC12 cells exposed to agonists (bradykinin, carbamylcholine, and ATP) binding to receptors coupled to polyphosphoinositide hydrolysis. With all the treatments employed, the response to an individual agonist was often incomplete, i.e., composed of either release from intracellular stores or influx only. In individual cells the responses were closely similar when only one and the same agonist was employed, and markedly heterogeneous, with considerable variation of the release/influx ratio, when different agonists were delivered in sequence. In a recently isolated PC12 cell clone, heterogeneity of the receptor-induced [Ca2+]i responses was markedly lower than in the overall population, although the release/influx ratio was still variable. We conclude that the large response heterogeneity observed in the overall PC12 cell population is due (a) to the coexistence of multiple clones; and (b) to the variable activation of intracellular transduction mechanisms.