Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils

Jennifer L. Bankers-Fulbright, Gail M. Kephart, Kathleen R. Bartemes, Hirohito Kita, Scott M. O'Grady

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5749-5757
Number of pages9
JournalJournal of Cell Science
Volume117
Issue number24
DOIs
StatePublished - Nov 15 2004

Fingerprint

Cytoplasmic Granules
Platelet Activating Factor
Eosinophils
Proton-Translocating ATPases
Interleukin-5
Cell Membrane
Ionomycin
Thapsigargin
Calcium-Transporting ATPases
Calcium Ionophores
Tetradecanoylphorbol Acetate
Protein Kinase C
Protons
Cytoplasm
Esters
Proteins
Calcium
Membranes

Keywords

  • Eosinophils
  • H-ATPase
  • IL-5
  • PAF
  • pH regulation

ASJC Scopus subject areas

  • Cell Biology

Cite this

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils. / Bankers-Fulbright, Jennifer L.; Kephart, Gail M.; Bartemes, Kathleen R.; Kita, Hirohito; O'Grady, Scott M.

In: Journal of Cell Science, Vol. 117, No. 24, 15.11.2004, p. 5749-5757.

Research output: Contribution to journalArticle

Bankers-Fulbright, Jennifer L. ; Kephart, Gail M. ; Bartemes, Kathleen R. ; Kita, Hirohito ; O'Grady, Scott M. / Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils. In: Journal of Cell Science. 2004 ; Vol. 117, No. 24. pp. 5749-5757.
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AB - 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.

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