The Role of Extracellular Calcium in the Neuropeptide-Y-Induced Increase in Cytosolic Calcium in Human Erythroleukemic (HEL) Cells

Abstract

Cytosolic calcium changes were followed in human erythroleukemic (HEL) cells loaded with the fluorescent probe fura-2. Peak increases in cytosolic calcium were reduced by two-thirds in cells suspended in Ca 2+-free medium, suggesting that calcium entry significantly contributes to the increases in cytosolic calcium after NPY receptor stimulation. To establish if Ca 2+ entry was a direct consequence of receptor stimulation or indirectly via depletion of Ca 2+ stores, the latter were totally or partially depleted by treatment with cyclopiazonic acid or α-thrombin, respectively, in Ca 2+-free medium. Partial depletion markedly diminished and full depletion suppressed the NPY-induced response in Ca 2+-free medium. After full depletion, the recovery of the NPY-induced increase in cytosolic calcium was dependent on the length of [Ca 2+] 3 reexposure, suggesting a direct entry of Ca 2+ to the storage sites followed by release to the cytosol. After partial depletion, transient reexposure to [Ca 2+] 3 did not by itself increase cytosolic calcium levels or refill the stores as NPY stimulation did not increase cytosolic calcium if [Ca 2+] 3 was chelated prior to stimulation. However, if partially depleted cells were exposed to NPY in the presence of readded [Ca 2+] 3, the peak calcium response was similar to that of control cells, indicating that partially depleted calcium stores can be refilled 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 depletion, while under physiological conditions calcium entry is coupled to receptor stimulation.