Relationship between time of activation of phospholipase C-linked plasma membrane receptors and reloading of intracellular Ca2+ stores in LAN-1 human neuroblastoma cells.

The effect of sequential stimulation of different inositol (1,4,5)-trisphosphate (IP3)-linked receptors on the functioning of intracellular Ca2+ stores was evaluated in single LAN-1 human neuroblastoma cells by means of fura-2 microfluorimetry. Homologous restimulation both in the absence and in the presence of extracellular Ca2+ with endothelin-1 (ET-1), Lys-bradykinin (BK), and ATP did not elicit an intracellular Ca2+ increase, whereas a [Ca2+]i elevation after carbachol (CCh) re-exposure was obtained only in the presence of extracellular Ca2+. Since thapsigargin and ionomycin, in the absence of extracellular Ca2+, were still able to release Ca2+ after ET-1, BK, and ATP but not after CCh, it can be argued that in the first case the stores were not completely depleted. This evidence was also confirmed by the fact that LAN-1 cells, sequentially exposed in different order to ET-1, BK, ATP, and upon extracellular Ca2+ removal, showed an increase of [Ca2+]i although progressively reduced in magnitude. By contrast, when CCh was perfused as the first agonist, it completely precluded any further Ca2+ mobilization by the other three agonists. In addition, the lack of potentiation of the Ca2+ response when BK and ET-1 were superfused together and the potentiation of Ca2+ response elicited by ET-1 after BK, when the plasma membrane Ca2+ efflux pathways were blocked by lanthanum during the first agonist exposure, indicated that LAN-1 cells can recycle cytoplasmic Ca2+ when exposed to ET-1, BK, ATP but not when exposed to CCh. This inhibitory effect of CCh (perfused for 90 s) on Ca2+ refilling was strictly dependent on the time of receptor occupancy since the exposure to CCh for a shorter period (15 s) produced the same effect on Ca2+ refilling when ET-1, BK, and ATP were perfused, as first agonist, for 90 s. Furthermore, the entity of Ca2+ refilling after 15 s of BK receptor occupancy was similar to that observed after 90 s. This seems to suggest that the receptors for ET-1, BK, and ATP maintain the transductional mechanisms in an activated state for a time shorter than the time of receptor occupancy. This was confirmed by the fact that IP3 levels during a 90-s BK exposure fell to prestimulated value within 30 s, whereas after CCh they reached a sustained plateau phase, after the peak.