Effects of recombinant human granulocyte-macrophage colony-stimulating
factor on intracellular pH in mature granulocytes
R Sullivan, JD Griffin, J Wright, DA Melnick, JL Leavitt, JP Fredette, JH Horne , CA Lyman, KG Lazzari and ER Simons
Department of Medicine, Boston City Hospital, MA.
We studied the effects of recombinant human granulocyte-macrophage
colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes
using the fluorescent probe BCECF. GM-CSFrh did not directly alter the
resting pH of granulocytes isolated from the peripheral blood; however,
when the cells were preincubated for 90 minutes with the growth factor and
then activated with the chemotactic peptide N-formyl met leu phe (fMLP),
they exhibited both an acceleration in the initial rate of acidification
and a marked delay in realkalinization. The kinetic changes both in initial
acidification and in subsequent realkalinization induced by GM-CSFrh
priming were not prevented by protein synthesis inhibitors and were
observed in granulocytes harvested from patients with both sex-linked and
autosomal recessive chronic granulomatous disease (CGD). By directly
quantitating H+ ion secretion, by monitoring the effects of sodium
repletion on intracellular pH, and through use of the sodium channel
inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor
ouabain, we showed that the altered kinetics of intracellular acidification
and alkalinization following fMLP stimulation of GM-CSFrh- primed
granulocytes could not be accounted for by changes in transmembrane proton
exportation regulated by the Na+/H+ antiport channel. Although the initial
acidification following fMLP was abrogated by 2-deoxy-D-glucose in both
GM-CSFrh-pretreated and GM-CSFrh- untreated granulocytes, retardation of
the subsequent phase of alkalinization was observed in GM-CSFrh-primed
cells even after inhibition of both glycolytic and mitochondrial
metabolism. Our data indicate that the increased cytosolic acidification
following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not
result from disordered proton excretion but instead from increased release
of intracellular free acid which is only partially coupled to glucose
catabolism or to the generation of superoxide anion (O2-).
Volume 72,
Issue 5,
pp. 1665-1673,
11/01/1988
Copyright © 1988 by The American Society of Hematology
