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.
Highlights
Eosinophils account for approximately 2-3% of peripheral white blood cells and are involved in clearing helminth infections
We report the effects of calcium mobilizing and protein kinase C (PKC) activating agents on pHi in isolated human eosinophils
The alkalinization response was not dependent on the activities of plasma membrane acid or base transport mechanisms and directly correlated with a decrease in intragranule pH. These results suggest that cytoplasmic alkalinization is a consequence of calcium-dependent activation of H+-ATPases localized in the granule membranes
Summary
Eosinophils account for approximately 2-3% of peripheral white blood cells and are involved in clearing helminth infections (reviewed by Giembycz and Lindsay, 1999). Several studies have shown that a voltage-dependent, Zn2+-sensitive plasma membrane proton channel is activated in parallel with the increase in NADPH oxidase activity (Cherny et al, 2001; Cherny et al, 2003; DeCoursey et al, 2001; DeCoursey et al, 2003; Gordienko et al, 1996; Petheo et al, 2003) This channel is regulated by mediators that increase PKCδ activity and, following PKC activation, the cytosolic pH (pHi) does not significantly change in response to increases in superoxide production (Bankers-Fulbright et al, 2001). Proton channels play a major role in the control of pHi in activated human eosinophils
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