Cholesterol-modulating agents selectively inhibit calcium influx induced by chemoattractants in human neutrophils.

ABSTRACT

The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-beta-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn(2+) induced by fMet-Leu-Phe or thapsigargin. Methyl-beta-cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10(-10) m platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10(-7) m platelet-activating factor. On the other hand, methyl-beta-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of FcgammaRIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-beta-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.