THE ROLE OF EXTRACELLULAR CALCIUM IN THE NEUROPEPTIDE-Y-INDUCED INCREASE IN CYTOSOLIC CALCIUM IN HUMAN ERYTHROLEUKEMIC (HEL) CELLS

Cytosolic calcium changes were followed in human erythroleukemic (HEL) cells loaded with the fluorescent probe fura-2. Peak increases in cyt osolic calcium were reduced by two-thirds in cells suspended in Ca2+-f ree medium, suggesting that calcium entry significantly contributes to the increases in cytosolic calcium after NPY receptor stimulation. To establish if Ca2+ entry was a direct consequence of receptor stimulat ion or indirectly via depletion of Ca2+ stores, the latter were totall y or partially depleted by treatment with cyclopiazonic acid or alpha- thrombin, respectively, in Ca2+-free medium. Partial depletion markedl y diminished and full depletion suppressed the NPY-induced response in Ca2+-free medium. After full depletion, the recovery of the NPY-induc ed increase in cytosolic calcium was dependent on the length of [Ca2+] (e) reexposure, suggesting a direct entry of Ca2+ to the storage sites followed by release to the cytosol. After partial depletion, transien t reexposure to [Ca2+](e) did not by itself increase cytosolic calcium levels or refill the stores as NPY stimulation did not increase cytos olic calcium if [Ca2+](e) was chelated prior to stimulation. However, if partially depleted cells were exposed to NPY in the presence of rea dded [Ca2+](3), the peak calcium response was similar to that of contr ol cells, indicating that partially depleted calcium stores can be ref illed from extracellular sources only if NPY receptors are stimulated. Analysis of the data suggests that in HEL cells the entry of calcium and mobilization from intracellular stores are in series processes and that entry is triggered by intracellular levels only under extreme de pletion, while under physiological conditions calcium entry is coupled to receptor stimulation. (C) 1994 Academic Press, Inc.