Activation of the Na /H antiport mechanism was studied. In human neutrophils by monitoring. Intracellular. pH wIth a carboxyfluorescein derivative.
Journal
Intracellular
pH Changes Na/H
Steven
J. Weisman,
Departments
Amalia
Biology
During Neutrophil Antiport
Punzo,
of Pediatrics (S.J.W., Connecticut
of Leukocyte
Charles
Ford,
A.P., CF.), and Physiology Health Center, Farmington
(1987)
Activation:
and Ramadan (R.l.S.),
41:25-32
University
I. Sha’afi of
Activation of the Na /H antiport mechanism was studied In human neutrophils by monitoring Intracellular pH wIth a carboxyfluorescein derivative. N-formyl-methlonylleucyl-phenylalanine (FMLP) and phosphollpase C (PLC) Induced blphaslc pH changes. Amiloride, which inhibits the antiport, completely blocked alkalinization but enhanced acidification. Polymyxin B, which Inhibits protein kinase C, only blocked alkalinization. Activation wIth phorbol 12-myristate 13-acetate (PMA) led to alkallnlzation only; this was Inhibited by amllorlde or polymyxln B. Thus, during polymorphonuclear leukocyte (PMN) activation, intracellular alkallnlzation appears to be mediated by an amlioride-sensitlve Na/H + antiport. Antiport activity can also be blocked Indirectly by Inhibition of protein kinase C activity. Early Intracellular acidification does not appear to require kinase activity but is observed when phospholipids are remodeled with PLC. The antiport was also activatable by hypertonic buffered media. This response did not appear to be mediated by protein kinase C because it was unaffected by polymyxin B. Finally, superoxide generation was investigated. It is affected by, but not soley controlled by, either antiport or protein kinase C activity. Key words:
protein
kinase
C, superoxide
INTRODUCTION The presence of a Nat/H + transport mechanism in neutrophils (PMN) has been demonstrated and it has been shown that the addition of the chemotactic factor Nformyl-methionyl-leucyl-phenylalanine lates
this
system
the
protein
(FMLP)
[9, 21, 23
kinase
,
25].
C (PKC)
It has
system
activation of the Na+/H + antiporter mous soluble stimuli in several cell
neutrophil experimental myristate
[27, 28]. This observation 13-acetate
PKC, activate the that this stimulation centrations has been
factor
insulin
activation
that
is involved
in the
mechanism by vartypes, including the
(PMA),
which
bind
to and
activate
Nat/H + antiporter mechanism is inhibited by relatively high [2,7, addition
to quiescent
of PKC
may
and con-
12, 20] Recently, it of epidermal growth .
cultures
cells failed to stimulate PKC but Nat/H + antiport in cells lacking Thus
stimu-
suggested
view is based on the common that agents such as phorbol 12-
of trifluoperazine found that the
plus
been
of Swiss
3T3
elicited activation the PKC system
not be the only
of the [26].
sequence
of
intracellular events leading to antiport stimulation. The present studies were undertaken to investigate the role of PKC in the activation of the Na/H antiport in human neutrophils. The results to be reported strongly suggest
that
stimulation
© 1987 Alan R. Liss,
of the
Inc.
Nat/H
+
transport
system
in these cells is mediated ways, only one of which
PKC
We also
.
by at least is dependent
provide
evidence
sient acidification seen vation is due to factors addition,
we
antiport
may
generating
play
the
mechanism
in
in the presence to inhibit PKC
MATERIALS Materials The
Received Reprint versity
PKC
and
the
Na /H
of the superoxide
these
cells.
in O peptide
release induced
of polymyxin [14,17,30].
(O)
Although
direct
and activation O release B (PB),
of can
which
reagents
were
obtained
from
is
Sigma
Co (St Louis, MO): cytochrome C (type dismutase, FMLP, Boc-Phe-Leu-Phe-Leu-Phe
(Boc-Phe), chased
role
pH iran-
peptide acticontrol. In
AND METHODS
following
Chemical superoxide
the early
chemotactic from antiport
in activation
activation of PKC results the antiport, chemotactic occur known
that
during distinct
investigated
two separate pathupon activation of
polymyxin from January
B, and
Consolidated 31,
1986;
nigericin.
Midland accepted
requests: Steven J. Weisman, of Connecticut Health Center,
July
Co 30,
Department Farmington,
PMA
was
(Brewster,
VI), purNY).
1986. of Pediatrics, CT 06032.
Uni-
26
Weisman
Phospholipase
et al
C was
obtained
from
Calbiochem
(La Jolla, 00-
CA). We obtained 2’ ,7 ‘-bis-(2-carboxyethyl)-5 ,6-carboxyfluorescein tetra-acetoxymethyl ester (BCECF-AM) from
Molecular
Probes
(Junction
City,
OR).
Hanks’
balanced
salt
solution
with
10 mM
out magnesium (pH 7.4), except periments as indicated below. Preparation
HEPES
‘C
and in a with-
for the superoxide
ex-
of Cells
Human neutrophils ized venous blood [3].
0
Amiloride
(AM) was the generous gift of the Merck, Sharpe Dohme Co (Rahway, NJ). Cells were suspended
Isolated
were prepared from fresh heparinaccording to the method of Boyum
PMNs
were
suspended
in the
Review
Board
and
according
to the
Helsinki
Declaration. Intracellular
80
I-
z
. 70-
I-
z LU
C) () LU
60
0
50
-J
40
appropriate
buffer at the concentrations specified below. All samples were collected from normal adult volunteers following approval of the University of Connecticut Health Center Institutional
(1)
pH (pH1)
7.5
7.0 pH
Fig. 1. pH Calibration. Neutrophils loaded with BCECF were suspended in various pH buffers. Extracellular and intracellular pH were equilibrated with nigericin (10 zM) and fluorescence was measured. Points were collected in two separate experiments.
BCECF-AM was dissolved in dried dimethyl sulfoxide Figure 1 represents the results of two typical at 0.5 mg/mi. Aliquots were stored dessicated at -70#{176}C. measured. calibration experiments demonstrating that there is a unPMN (2 x 107/mi) were loaded with 2’,7’-bis-(2-carboxear relationship between fluorescent intensity and pH yethyl)-5,6-carboxyfluorescein (BCECF) by incubation with the tetra-acetoxymethyl ester (3.65 m) for 30 mm over the range studied (R = .988; P < .0001 for the linear regression). In four separate calibration experiments, at 37#{176}C. Impermeant BCECF is generated by the action resting pH1 of unstimulated PMN was 7.26± .02. of cytoplasmic esterases [13] Fluorescent measurements were made in an SLM 8000 spectrofluorimeter (SLM .
Instruments, lengths
both
Urbana, were
slit widths
was equipped stirring magnetic
at 37#{176}C during dardized
to
IL).
Excitation
and
emission
wave-
500 nm and 530 nm, respectively, while were set at 4 nm. The cuvette chamber with a micro-injection port and a constantflea
bar.
Temperature
was
Gain
settings
all experiments. permit
maintained
were
experiment-to-experiment
stan-
compar-
isons.
BCECF-loaded as measured trypan blue.
PMNs
demonstrated
>
by the ability to exclude Fluorescent dye appeared
tributed in the were examined
cytoplasm of the PMNs by fluorescence microscopy.
BCECF-loaded
PMNs
cells
when
released
stimulated
with
O
PMA
95 % viability
the supravital to be evenly when
was
calibrated
over
7.8 with the method of Moolenaar suspended in Nat-free Hanks’ which isotonicity was maintained phore external
mgericin
recorded
and
and
suspension
KOH
or
(10 internal the
HC1
either
equilibrated
then
the
added to Fluorescent pH
of the
pre-incubated
trated
solutions
cient
to increase
range
of 6.4-
et al [13]. PMN were balanced salt solution in with KC1. The pH was
and
zM) was pH [19].
the pH
was
to control
total).
K/H equilibrate intensity suspension
ionothe
was was
for 5 mm in the 1 x 107/mi. The
PMN were incubated at a cell density of stimulated
with
PMA,
FMLP,
or
PLC. In other experiments, AM (1 mM) or PB (0.4 mg/ mi) was allowed to pre-incubate with the cells for 10 mm before the addition of the various stimuli. Alternatively, PB or AM were added to the spectrophotometric cuvette 12 mm after stimulation with FMLP when the alkalimzation plateau had stabilized. Boc-Phe (l0 M) was phase
posed
200
Fluorescence
with
cell
teau
pH Calibration
adjusted
BCECF-loaded spectrofluorimeter
the cells In addition,
similarly
(>90%
dye dis-
pH Experiments
with
cells
and responses
to hypertonic
buffers
of NaCl, the
or added
measured. by choline
extracellular
during
Cells the
addition
the
were
pla-
also
of
ex-
concen-
chloride,
or urea
buffered
osmolarity
suffiby
mOsm.
Superoxide
Release
The continuous
measurement of O-dependent cytochrome C reduction was performed at 37#{176}C in a Lambda 3B double-beam spectrophotometer equipped with a 3600 Data Station (Perkin-Elmer, Norwalk, CT) using the method PMNs phosphate
of Cohen and were suspended with
glucose
Chovaniec [5]. Briefly, isolated at 1 x l06/mi in Krebs-Ringers(pH
7.3)
plus
119 M
cytochrome
Intracellular C. The reference oxide dismutase.
cuvette contained 0.02 mg/mi of superThe various stimuli were introduced
pH in PMN:
Na’/H
Antiport
27
020
simultaneously to both the reference and experimental cuvettes and the reduction of cytochrome C was followed spectrophotometrically at 550 nM. Preincubations for 10
015
A B
010
mm with amiloride (1 mM) or PB (0.4 mg/mi) were performed as indicated below The release of O was determined as the change in absorbance from baseline until the reduction of cytochrome C reached a plateau or after 10 mm. This was converted to nanomoles of re-
005
.
duced 21.1
x
cytochrome 103molcm’.
Statistical
C by use
of the
extinction
analysis
RESULTS pH1 Changes
units
over below
curve
for
work of Molski et al, or l06M induced a
pH1 changes
(Fig.
baseline
was
reached.
This
acid
and then pH continued
alkalinization with 100
virtually
[10].
2
3
4
5
6
7
(mm)
TIME
Fig. 2. Intracellular pH changes. pH1 changes are shown for stimulation by 100 ng/ml PMA (A), 107M FMLP (B), and 0.75 x 10-6 M PLC (C). Each tracing represents the average of 7, 16, and 3 experiments, respectively. Stimuli were added at time 0.
when PMN stimulation ng/mi PMA (Fig. 2A).
pH1 changes (0.75 x l06
identical
to that
were
allowed bation
measured
a uni-
was accomOn the other
after
mains
in significant
under
the
FMLP with
acidification
in either
unstim-
or in cells stimulated by PMA. to assess if the plateau of alkalinization
AM.
reaction
cuvette
a brief ibrated
lag period, to levels
dification. Thus pears to be under
influence
of the
activation After during
Na/H
to proceed 12 mm, the
alkaline
added plateau
pH promptly fell that were comparable maintenance the control
without
we
re-
antiport,
and
we
pre-incuAM
to
phase.
the
After
ultimately equilto the initial aci-
of pH1 alkalinization of the Na /H + antiport.
ap-
introduc-
M), the response pattern seen with FMLP (Fig. 2C).
pH dropped by 0.037±0.013 units, followed mate alkalinization of 0.53 ±0.013 units.
to result
ulated cells In order
re-
phase
by
appear
depH
to increase until (0. 104 ±0.045
a plateau of alkalinization was reached units). This biphasic response was replaced
hand, when tion of PLC
2B)
of chemotactic peptide, pH1 rapidly 40-50 5 until a nadir of 0.056±0.017
to baseline
phasic pushed
I
With PMN Activation
response
addition
turned
-005
-0
package.
As predicted from the previous FMLP at concentrations of i07
creased
0
Methods
statistical
After
Q.
coefficient
When applicable, data were analyzed with the Student’s t-test. In addition, data were analyzed by regression analysis using an Apple II series computer and a
biphasic
I
was The
by an ultiA
Effect of Amiloride Cells
incubated
on PHI in AM
for the observation ulated
period
experiments
(data
alone
maintained
employed not
baseline
in the standard
shown).
Prior
pH1 stim-
addition
I
0.
B
of
AM completely prevented alkalinization for either FMLP, PLC, or PMA (Figs. 3C, 4C, SC). Furthermore, AM enhanced acidification during stimulation with both FMLP and PLC. Stimulation with FMLP led to 0.076±0.12 PLC were
pH units
acidification,
led to 0.106±0.010 both significantly
units different
while change. from
stimulation
with
These changes the acidification
seen in control experiments tively). Thus the alkaline actmvatmon appears to be
(P < .05 and
