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J Biol Chem, Vol. 274, Issue 37, 26098-26104, September 10, 1999
From the Cell Biology Programme, Research Institute, The Hospital
for Sick Children, Toronto, and the Department of Biochemistry,
University of Toronto, Toronto, Ontario M5G 1X8, Canada
The electrogenic activity of the
NADPH oxidase is associated with depolarization of the plasma membrane
in activated neutrophils. The magnitude and consequences of this
depolarization, however, remain unknown. Neutrophils are not amenable
to electrophysiological determinations of membrane potential by current
clamp. Instead, the occurrence of depolarization has been inferred from
the use of potential-sensitive fluorescent dyes. However, such dyes
partition into intracellular organelles and may yield erroneous
results, particularly because the NADPH oxidase resides largely in
secretory granules, where it has been claimed to become activated. We
confirmed the intracellular generation of oxidase products using
dihydrorhodamine, which is converted to the fluorescent rhodamine 123 when oxidized. Rhodamine 123 accumulated inside endomembrane organelles
in both neutrophils and in differentiated HL60 cells, where it
co-localized with the primary granule marker CD63. To estimate the
surface membrane potential without interference from organelles, we
devised a method based on the voltage-driven uptake of
Mn2+ across the plasmalemma. The uptake of
Mn2+ through calcium release-activated channels was
measured as the rate of Indo-1 fluorescence quenching in
thapsigargin-treated cells. The rate of Mn2+ influx was
found to vary when the membrane potential was manipulated using
conductive ionophores and also when the NADPH oxidase was activated. A
calibration curve in the positive potential range was constructed using
the Na+ ionophore SQI-Pr. Using this calibration, the
membrane potential of phorbol ester-activated neutrophils was found to
reach +58 ± 6 mV, a sustained depolarization of over 100 mV
compared with the resting potential. The depolarization was greatly
diminished when the NADPH oxidase was inhibited with diphenylene
iodonium. Together, these results indicate that the NADPH oxidase can
generate a large depolarization of the plasmalemma, which should
suffice to activate a variety of voltage-gated channels, including the outwardly rectifying H+ conductance.
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