Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils.
The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1+/-0.04 to 7.5+/-0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.
Bankers-Fulbright
Jennifer L
JL
Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Kephart
Gail M
GM
Bartemes
Kathleen R
KR
Kita
Hirohito
H
O'Grady
Scott M
SM
D000251:Adenosine Triphosphatases / Q000737:chemistry; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D047091:Eosinophil Granule Proteins / Q000502:physiology*; D047093:Eosinophil Major Basic Protein / Q000737:chemistry; D004804:Eosinophils / Q000166:cytology / Q000378:metabolism*; D004952:Esters; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D015848:Interleukin-5 / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis; D010972:Platelet Activating Factor / Q000737:chemistry / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D047091:Eosinophil Granule Proteins; D004952:Esters; D005452:Fluoresceins; D015848:Interleukin-5; D010972:Platelet Activating Factor; D011522:Protons; D012333:RNA, Messenger; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D019284:Thapsigargin; D011493:Protein Kinase C; D047093:Eosinophil Major Basic Protein; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D015032:Zinc; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium