Sustained Depolarization and ADP-Ribose
Activate a Common Ionic Current in Rat
Peritoneal Macrophages
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J Immunol 2003; 170:1167-1173; ;
doi: 10.4049/jimmunol.170.3.1167
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References
Brice Campo, Annmarie Surprenant and R. Alan North
The Journal of Immunology
Sustained Depolarization and ADP-Ribose Activate a Common
Ionic Current in Rat Peritoneal Macrophages1
Brice Campo, Annmarie Surprenant, and R. Alan North2
Phagocytosis is associated with large changes in the membrane potential of macrophages, but the functional significance of this
is unknown. Whole cell recordings were made from rat peritoneal macrophages. Sustained (>30 s) depolarization of the cells
progressively activated a conductance that remained high (several nanoSeimens) for several tens of seconds. This current: 1) was
linearly dependent on potential between ⴚ100 and ⴙ50 mV; 2) reversed close to 0 mV in a physiological external solution; 3) could
also be carried in part by N-methyl-D-glucamine (PNMDG/PNa 0.7), chloride (PCl/PNa 0.5), or calcium (PCa/PNa 1.3); and 4) was
blocked by intracellular ATP (5 mM) or ADP (10 mM) and by extracellular lanthanum (half-maximal concentration 1 mM). A
current with all the same properties was recorded in cells when the intracellular solution contained ADP-ribose (10 –300 ␮M) or ␤-NAD
(1 mM) (but not any other nucleotide analogs tested). The results suggest that prolonged depolarization leads to an increased intracellular level of ADP-ribose, which in turn activates this nonselective conductance(s). The Journal of Immunology, 2003, 170: 1167–1173.
Institute of Molecular Physiology, University of Sheffield, Sheffield, United Kingdom
Received for publication July 9, 2002. Accepted for publication November 18, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Wellcome Trust.
2
Address correspondence and reprint requests to Dr. R. Alan North, Institute of
Molecular Physiology, Alfred Denny Building, Western Bank, University of Sheffield, Sheffield S10 2TN, U.K. E-mail address: [email protected]
Abbreviations used in this paper: ␤NAD⫹, ␤-NAD free acid; ␤NADH, ␤-NAD disodium salt; ␤NADP⫹, ␤-NAD phosphate disodium salt; ␤NADPH, ␤-NADPH disodium
salt; NMDG, (N-methyl-D-glucamine); SBFI, 1,3-benzenedicarboxylic acid, 4,4⬘-[1,4,10trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetra-ammonium salt; SKF96365, (1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride.
1 mM) (12, 13) and might therefore underlie the current following
phagocytosis described by Holevinsky and Nelson (1). This current is also activated by intracellular ADP-ribose at concentrations
(10 –300 ␮M) lower than those required for ␤NAD⫹. It corresponds in some of its properties to the currents observed with
heterologous expression of TRPM2 subunits (formerly called
LTRPC2; for nomenclature, see Ref. 14) (12, 13, 15).
These results are consistent with the interpretation that the prolonged depolarization that follows phagocytosis described by
Holevinsky and Nelson (1) results from an inward current, such as
that activated by ␤NAD⫹ in U937 cells (12, 13). However, during
the course of voltage-clamp recordings from rat peritoneal macrophages, we noticed that a sustained inward current could actually
be activated by prolonged depolarization. In this work, we describe
some properties of this current, and compare these with the current
activated by ␤NAD⫹ and ADP-ribose in the same cells. Both currents showed the same permeability to sodium, calcium, N-methylD-glucamine (NMDG), chloride, and gluconate ions, and sensitivity to
block by lanthanum. The current activated by intracellular ADP-ribose occluded the depolarization-activated current. Thus, we conclude it is likely that these two stimuli activate the same conductance.
Materials and Methods
Culture of rat peritoneal macrophages
Rat peritoneal cells were obtained from male Wistar rats (10 wk old) killed
by cervical dislocation. The peritoneal cavity was lavaged with PBS (Life
Technologies Cell Culture, Invitrogen, Paisley, U.K.). After centrifugation,
the macrophage fraction was separated from the mast cells by fluorescence-activated cell sorting. Two populations of cells were separated by
forward and side scatter. Cells were then collected by centrifugation and
resuspended in DMEM (Life Technologies Cell Culture) containing 10%
(v/v) FCS (Sigma-Aldrich, Poole, U.K.), 1% glutamine (Calbiochem, La
Jolla, CA), and 1% antibiotics (Life Technologies Cell Culture, Invitrogen). Cells were plated onto glass coverslips and kept at 37°C in a humidified atmosphere containing 5% CO2. The cultured macrophages were used
in patch-clamp experiments 1–3 days after plating. Cells were identified as
macrophages using immunohistochemistry staining with the macrophage
specifics Abs ED1–10 and ED-2 (Serotec, Oxford, U.K.).
HEK293 cells expressing P2X2/3 receptors
3
Copyright © 2003 by The American Association of Immunologists, Inc.
HEK293 cells were transfected, as previously described (16). Briefly, cells
were electroporated (Gene Pulser; Bio-Rad, Hercules, CA) in the presence
of 30 mg of plasmid containing both P2X2 and P2X3 receptor cDNAs.
Following electroporation, cells were plated and selected for neomycin
resistance, subcloned by FACS sorting, and resuspended in standard
0022-1767/03/$02.00
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T
he membrane potential of macrophages undergoes a prolonged depolarization following phagocytosis of an immune complex (1). This depolarization follows an increase in superoxide by a variable period, but typically tens of
seconds, and it is subsequently maintained for several minutes.
The underlying current shows little rectification and reverses close
to 0 mV. The current is blocked by an inhibitor of NADPH-oxidase, and mimicked by xanthine and xanthine oxidase, leading to
the conclusion that the depolarization results from free radical release within the cell following particle engulfment. This finding is
in keeping with a body of work in a range of cells, which indicates
that oxidative stressors, such as H2O2, can activate inward currents
(2, 3). The capacity of such currents to deliver calcium to the cell
has been considered to contribute to subsequent oxidative damage.
Although intracellular calcium plays a key role in several aspects of macrophage function, including activation, migration, and
proliferation as well as generation of superoxide anion and stimulation of NO production, calcium influx is not immediately required for phagocytosis (4 – 6). It is unlikely that the well-studied
store-operated or capacitative calcium entry current, which is
present in all nonexcitable cells, could underlie the current associated with phagocytosis in macrophage because it shows a characteristically strong rectification, the currents are very small (only
a few pA at ⫺60 mV), and it is relatively impermeable to sodium
under normal conditions (6 –11). Recently, a current has been described in the human macrophage U937 cell line that is activated
by extracellular H2O2, or intracellular ␤-NAD free acid (␤NAD⫹;3
1168
growth medium (Dulbecco’s) supplemented with 10% heat-inactivated
FCS and 2 mM L-glutamine. Cells were grown to confluency and plated on
coverslips, and recordings were made 2– 48 h later.
Whole cell recording
Intracellular sodium measurement
Cells were loaded with 1,3-benzenedicarboxylic acid, 4,4⬘-[1,4,10-trioxa7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetra-ammonium salt (SBFI, 10 mM; Molecular Probes, Eugene, OR), and
0.02% pluronic acid for 45 min at 37°C. Ratiometric fluorescence measurements were performed using an Axiovert 100 microscope (Zeiss,
Oberkochen, Germany). Cells were excited at 340 and 380 nm using a
computer-driven monochromator, and the emission fluorescence (510 nm)
was collected through a bandpass filter onto a charge-coupled device camera. Data were analyzed with Photonics software (Planegg, Germany).
Measurements of permeability
We first measured the permeability of chloride relative to that of sodium
(PCl/PNa) in bi-ionic conditions. PCl/PNa was then calculated from
PCl/PNa ⫽ ((1 ⫺ exp(⫺x)) ⫻ ([Na]o ⫺ [Na]i exp(x)))/(([Cl]i exp(⫺x) ⫺
[Cl]o) ⫻ (1 ⫺ exp(x))) (equation 1), where x ⫽ Er F/RT (extracellular 0.3
mM calcium ignored), where Er is the reversal potential, R is the Gas
constant, F is the Faraday, and T is the absolute temperature. We used this
value of PCl/PNa in subsequent calculations in which three permeant ions
were present. The expressions used were: PNMDG/PNa ⫽ ((([Na]i exp(x) ⫺
[Na]o)/(1 ⫺ exp(x))) ⫹ PCl/PNa ⫻ (([Cl]i exp(⫺x) ⫺ [Cl]o)/(1 ⫺
exp(⫺x))))/(([NMDG]o ⫺ [NMDG]i exp(x))/(1 ⫺ exp(x))) (equation 2);
Pgluc/PNa ⫽ ((([Na]i exp(x) ⫺ [Na]o)/(1 ⫺ exp(x))) ⫹ PCl/PNa ⫻ (([Cl]i
exp(⫺x) ⫺ [Cl]o)/(1 ⫺ exp(⫺x))))/(([gluconate]o ⫺ [gluconate]i exp(⫺x))/
(1 ⫺ exp(⫺x))) (equation 3); and PCa/PNa ⫽ (([Na]i exp(x)/(1 ⫺ exp(x))) ⫺
PCl/PNa ⫻ (([Cl]o ⫺ [Cl]iexp(⫺x))/(1 ⫺ exp(⫺x))))/(4 [Ca]o/(1 ⫺ exp(2x))
(equation 4). Although not shown in these equations, we have multiplied
ion concentrations by the following activity coefficients: Na⫹, 0.8; K⫹, 0.8;
Cl⫺, 0.8; NMDG⫹, 0.8; gluconate⫺, 0.8; Ca2⫹, 0.28 (19).
Data analysis
The results shown in the figures are mean ⫾ SEM. Curve fits of the pooled
data were performed using Kaleidagraph (Synergy Software, Reading, PA)
and Excel (Microsoft, Redmond, WA) software. The lanthanum inhibition
curve was fitted with I/Imax ⫽ 100 (IC50n/(IC50n ⫹ [La]n)), where I is the
current at a given lanthanum concentration expressed as percentage of the
current in the absence of lanthanum (Imax), IC50 is the lanthanum concentration at which I ⫽ 0.5 ⫻ Imax, and n is the Hill coefficient. Concentrationcurrent curves were fitted with I/Imax ⫽ 100 ([ADP-ribose]n/([ADP-ribose]n ⫹ EC50n)), where I is the current activated by a given concentration
of ADP-ribose and expressed as percentage of the maximal currents (Imax),
EC50 is the ADP-ribose concentration evoking half of the maximal current,
and n is the Hill coefficient.
Chemicals
ADP, ADP-glucose, ADP-ribose, cyclic ADP-ribose, AMP, ATP, EGTA,
lanthanum chloride, D-ribose, D-ribose 5-phosphate, NMDG, xanthine, and
xanthine oxidase were purchased from Sigma-Aldrich. ␤NAD⫹, ␤-NAD disodium salt (␤NADH), ␤-NAD phosphate disodium salt (␤NADP⫹),
␤-NADPH disodium salt (␤NADPH), and 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (hydrochloride; SKF96365)
were purchased from Calbiochem. SBFI was from Molecular Probes.
Results
Macrophages had a capacitance of 11.4 ⫾ 0.4 pF (n ⫽ 110). The
cell diameter was 9.1 ⫾ 0.2 ␮m (n ⫽ 30). Step depolarizations
from a holding potential of ⫺80 mV (10 mV increments to ⫹40
mV, 500 ms duration) revealed no time-dependent currents when
a sodium chloride-based internal solution was used. When a potassium chloride-based solution was used (150 mM KCl, 10 mM
HEPES, 10 mM EGTA), the resting potential of the cells was
⫺30.9 ⫾ 0.7 mV (n ⫽ 11).
Current activated by depolarization
Cells held at ⫺80 mV showed no change in holding current or
conductance during 5–10 min of whole cell recording. However, if
the cell was held at ⫹10 mV for several tens of seconds and then
returned to ⫺80 mV, a large inward current was observed (Fig.
1A). This current quickly (⬍5 s) reached its peak amplitude (several hundred pA) and then declined during the next few minutes
(Fig. 1A; in 10 cells the amplitude after 2 min was 19 ⫾ 4% of the
peak amplitude). The duration of the depolarization required to
evoke the subsequent inward current was not studied in detail;
however, a depolarization to ⫹10 mV for only 10 s was not effective (n ⫽ 5). After an interval of several minutes, a second
depolarization again elicited a large inward current with similar
properties (n ⫽ 10).
The inward current was associated with a large increase in cell
conductance. The current-voltage relation of the cell remained essentially linear, and reversed close to 0 mV (⫺3.1 ⫾ 0.24 mV, n ⫽
18) in normal external solution (Fig. 1, B and C) (Table I). The
time course of the conductance increase was followed by holding
the cell at ⫹10 mV, but repeatedly applying brief ramp voltage
commands (⫺130 to ⫹50 mV in 500 ms; 5 s intervals). Fig. 1C
shows the time course of the resulting conductance increase. The
current (at ⫺80 mV) grew more slowly and was much smaller with
a sustained holding potential of ⫺40 mV (Fig. 1D), and it was
larger with depolarization to ⫹40 mV.
In low chloride extracellular solution (40 mM NaCl), the current
at ⫺80 mV was reduced to 51 ⫾ 16% (n ⫽ 3) of its control value;
the residual current reversed polarity at ⫺13.1 ⫾ 1.6 mV (n ⫽ 4)
(Fig. 2A); from equation 1 this corresponds to PCl/PNa of ⬃0.5
(Table I). When extracellular sodium was substituted by NMDG,
the current evoked by depolarization was reduced to 79 ⫾ 2% (n ⫽
6) of its control value (measured at ⫺80 mV). The residual current
reversed polarity at ⫺5.4 ⫾ 0.45 mV (n ⫽ 7) (Table I; Fig. 2A),
which indicates that NMDG is significantly permeable (PNMDG/
PNa 0.74; from equation 2). When extracellular gluconate replaced
chloride, the current at ⫺80 mV was reduced to 91 ⫾ 4% (n ⫽ 4),
and when extracellular calcium replaced sodium, the inhibition
was to 48 ⫾ 6% (n ⫽ 6) of control (Fig. 2B). By measuring the
reversal potential of the residual current (Table I), it was evident
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Ion currents were studied using the whole cell configuration of the patchclamp technique, as described by Hamill et al. (17). Glass electrodes (Harvard Apparatus, Edenbridge, U.K.) ranged from 6 to 8 M⍀ in resistance
and were fire polished. The indifferent electrode was an Ag-AgCl electrode
connected to the bath through a short 3 M KCl agar bridge. All the experiments were performed at room temperature using an EPC9 patch-clamp
system (HEKA, Lambrecht, Germany). The cells were kept in an extracellular solution containing (mM): NaCl, 147; KCl, 2; MgCl2, 1; CaCl2, 2;
HEPES, 10; and glucose, 13. The pH was adjusted to 7.3 by adding NaOH
(4 mM). The standard intracellular solution contained (mM): NaCl, 148;
HEPES, 10; EGTA, 10. We used intracellular sodium rather than potassium in most experiments so that voltage-dependent potassium currents did
not complicate our estimates of reversal potential. We used a high EGTA
concentration so as to reduce any involvement of store-operated conductances in those cases in which we had calcium-free extracellular solutions.
The pH was adjusted to 7.3 by adding NaOH (24 mM). Drug application
and solution exchanges were conducted using a fast flow RSC 200 system
(Biologic Science Instruments, Claix, France). Cells were held at ⫺80 mV,
except where stated, and current-voltage plots were obtained from ramp
commands (⫺130 to 50 mV in 500 ms). Permeability experiments were
conducted using standard intracellular solution and extracellular solution of
different composition. The low NaCl solution contained (mM): NaCl, 40;
HEPES, 10; CaCl2, 0.3 mM; glucose, 236 (pH brought to 7.3 with 4.2 mM
NaOH). The NMDG solution contained (mM): NMDG, base 154; HEPES,
10; glucose, 23 (pH brought to 7.3 with HCl; final Cl concentration 146
mM). The gluconate solution contained (mM): Na-gluconate, 147; HEPES,
10; glucose, 33 (pH brought to 7.3 with 4 mM NaOH). The isotonic calcium solution contained (mM): CaCl2, 110; Ca(OH)2, 2; HEPES, 10; glucose, 12 (pH 7.3). Series resistance was compensated by ⬃75%.
Liquid junction potentials were calculated directly from ionic mobilities
(18). Measured potentials were corrected for the junction potential existing
at the recording pipette before seal formation (pipette solution ⫺1.9 mV
with respect to bath solution) and for any change in junction potential at the
indifferent electrode when the bath solution was changed (⬍4 mV in all
cases).
IONIC CURRENTS IN MACROPHAGES
The Journal of Immunology
1169
that the conductance activated by prolonged depolarization is significantly permeable to chloride and gluconate, NMDG, and calcium ions. In each of these ion substitutions, the current-voltage
relation remained essentially linear (Fig. 2).
We conducted control measurements of the permeability of P2X
receptors expressed in HEK293 cells, using the same intracellular
and extracellular solutions. We used the agonist ␣␤meATP (30
␮M) to activate the inward current in cells expressing P2X2/3 subunits, which was typically ⬃1 nA in amplitude (20). We observed
that the agonist-induced current reversed polarity at 0.8 ⫾ 0.5 mV
(n ⫽ 23) in control extracellular solution, ⫺33 ⫾ 1.4 mV (n ⫽ 4)
in 40 mM NaCl, and ⫺86.5 ⫾ 0.4 mV (n ⫽ 4) in NMDG solution.
These reversal potentials correspond (from equations 1 and 2) to
relative permeabilities of PCl/PNa ⬍ 0.02 and PNMDG/PNa ⬍ 0.03
(see Ref. 21).
The current induced by depolarization was substantially blocked
by lanthanum (10 ␮M) (Fig. 1); the IC50 for this action was ⬃1
␮M (Fig. 3A). The current was also significantly inhibited (76 ⫾
10%, n ⫽ 4) by SKF96365 (100 ␮M); at 10 ␮M SKF96365 inhibited the current in some (75 ⫾ 8%, n ⫽ 4), but not other (n ⫽
3) cells. SKF96365 has been widely used to block nonselective
cation channels and store-operated calcium entry in nonexcitable
cells since its introduction by Merritt et al. (22). The current
evoked by depolarization was also greatly reduced by including in
the pipette either ATP (5 mM, inhibited by 89 ⫾ 5%, n ⫽ 4) or
ADP (10 mM, inhibited by 97 ⫾ 4%, n ⫽ 5) (Fig. 3B).
Current activated by ADP-ribose
An inward current progressively developed over tens of seconds in
cells held at ⫺80 mV, when the recording electrode contained
Table I. Reversal potentials and relative permeabilities for two macrophage currentsa
Depolarization
Control
Low chloride
NMDG
Gluconate
Isotonic calcium
a
ADP-Ribose
Remaining Current (%)
Erev (mV)
PX/PNa
Remaining Current (%)
Erev (mV)
PX/PNa
51 ⫾ 16 (3)
79 ⫾ 2 (6)
91 ⫾ 4 (4)
48 ⫾ 6 (6)
⫺3.1 ⫾ 0.24 (18)
⫺13.1 ⫾ 1.6 (4)
⫺5.4 ⫾ 0.45 (7)
⫺8.8 ⫾ 0.6 (6)
0.20 ⫾ 0.50 (6)
0.47
0.74
0.96
1.33
69 ⫾ 2 (4)
75 ⫾ 2 (4)
84 ⫾ 4 (4)
42 ⫾ 6 (3)
⫺2.7 ⫾ 0.65 (11)
⫺13.5 ⫾ 1.2 (4)
⫺4.8 ⫾ 0.35 (4)
⫺8.3 ⫾ 0.34 (4)
0.36 ⫾ 1.6 (5)
0.45
0.84
0.90
1.15
Mean ⫾ SEM for number of cells in parentheses.
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FIGURE 1. Current activated by depolarization of cell membrane. A, Example in one cell of current activated by sustained depolarization (⫹10 mV).
When the holding potential is returned to ⫺80 mV after 30 s depolarization, a large inward current develops and then progressively declines during the
next minutes. The current remaining after 90 s is quickly and completely blocked by lanthanum (10 ␮M). B, Current-voltage plots at the times indicated
in A, before, during, and after activation of the current. C, Current-voltage plots recorded at different times after moving to a holding potential of ⫹10 mV.
A voltage ramp (⫺130 to 50 mV in 500 ms) was applied at 5 s intervals (time indicated beside selected traces). D, Time course of development of current
at ⫺80 mV, taken from experiments such as those in C. A large current was evoked by holding at ⫹10 mV (F); this was smaller when the potential was
held at ⫺40 mV (Œ), and did not occur with a holding potential of ⫺80 mV (E). Points are mean amplitude (⫾SEM) for eight (⫹10 mV), three (⫺40
mV), and five (⫺80 mV) cells.
1170
ADP-ribose (Fig. 4A). The current-voltage relation remained linear
throughout this period, and reversed close to 0 mV (⫺2.7 ⫾ 0.6
mV, n ⫽ 11) (Fig. 4B). With 1 ␮M ADP-ribose, the development
of the current was fitted by an exponential with a time constant of
⬃1 min (Fig. 4C). The lowest concentration of ADP-ribose that
was effective was 100 nM, and maximum value of the current was
reached with ⬃100 ␮M; the EC50 was ⬃10 ␮M (Fig. 4D). Changing the level of intracellular calcium buffering did not obviously
alter the effectiveness of ADP-ribose to activate this current. In our
control, strongly buffered solution (10 mM EGTA, 0 mM calcium,
⬃1 nM [Ca]i) ADP-ribose (300 ␮M) elicited a current of 703 ⫾
119 pA (n ⫽ 3); with a higher level of intracellular calcium (11
mM EGTA, 2 mM calcium, ⬃100 nM [Ca]i), this was 811 ⫾ 149
pA (n ⫽ 5). No similar current developed when the recording
electrode contained ADP (100 ␮M), ADP-glucose (100 ␮M), cyclic ADP-ribose (100 ␮M), AMP (100 ␮M), ATP magnesium salt
(100 ␮M), D-ribose (100 ␮M), D-ribose 5-phosphate (100 ␮M),
␤NADH (300 ␮M or 1 mM), ␤NADP⫹ (300 ␮M), or ␤NADPH
(300 ␮M) (n ⫽ 3 cells in all cases). However, ␤NAD⫹ at a concentration of 1 mM (n ⫽ 4) evoked a current similar to that observed with ADP-ribose, although 100 and 300 ␮M were ineffective. The peak current elicited by ␤NAD⫹ (1 mM) was 758 ⫾ 428
pA (n ⫽ 4), which is not different from that seen with ADP-ribose
(Fig. 4).
The relative permeability of the current induced by ADP-ribose
(300 ␮M) was examined by measuring the reversal potential in
different extracellular solutions. From the values given in Table I,
it can be seen that the relative permeability to chloride, NMDG,
FIGURE 3. Similar inhibition of depolarization activated and ADP-ribose-activated currents. A, Dose-response curves for inhibition by lanthanum of the currents in macrophages evoked by depolarization (F, n ⫽ 3)
and ADP-ribose (E, n ⫽ 4). Recordings made at ⫺80 mV. (No difference
between depolarization and ADP-ribose: ANOVA, p ⬎ 0.05.) B, Currents
evoked by depolarization (filled bars) and by intracellular ADP-ribose
(open bars) have similar sensitivities to inhibition by intracellular ATP (5
mM) and ADP (10 mM). (No difference between depolarization and ADPribose: ANOVA, p ⬎ 0.05.)
gluconate, and calcium ions did not differ markedly from those
determined for the currents elicited by depolarization.
The current evoked by ADP-ribose was also very sensitive to
block by extracellular lanthanum (Fig. 3A), with an IC50 of ⬃10
␮M. It was also blocked by SKF96365 (100 ␮M, 77 ⫾ 4%, 5 of
5 cells tested; 10 ␮M, 70 ⫾ 7%, 7 of 12 cells tested) and by
intracellular either ATP (5 mM, inhibited by 66 ⫾ 11%, n ⫽ 5) or
ADP (10 mM, inhibited by 76 ⫾ 3%, n ⫽ 4) (Fig. 3B). These
relative inhibitions were not different from those observed for the
current activated by depolarization (ANOVA, p ⬎ 0.05).
A membrane current with similar properties was also elicited by
applying xanthine (0.1 mM) and xanthine oxidase (0.05 U/ml), to
generate superoxide anions; this current was completely blocked
by 100 ␮M lanthanum (Fig. 5A). Application of xanthine alone
had no effect. In parallel experiments, we used intracellular sodium
imaging as an index of depolarization of undisturbed macrophages.
In these cells, H2O2 (10 mM) evoked a progressive increase in intracellular sodium concentration ([Na]i) (Fig. 5B). The time course of the
[Na]i increase was similar to that of the inward current observed with
xanthine/xanthine oxidase, or depolarization, and the increase in [Na]i
was also strongly reduced by lanthanum (1 mM) (Fig. 5B).
Occlusion of the two currents
The similar properties of the two currents led us to hypothesize
that sustained depolarization and intracellular ADP-ribose were
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FIGURE 2. Relative permeability of conductance activated by prolonged depolarization. A, Current-voltage plots (ramp from ⫺130 to 50 mV
in 500 ms) in control solution, and in low (40 mM) sodium chloride, and
NMDG-chloride solutions. Reversal potentials are ⫹2, ⫺11, and ⫺5 mV
(see enlarged inset). B, Current-voltage plot from a different cell shows no
change in reversal potential when external solution is changed to isotonic
calcium chloride. Reversal potentials in this cell are 0 and ⫺2 mV for
control and isotonic calcium, respectively.
IONIC CURRENTS IN MACROPHAGES
The Journal of Immunology
1171
activating the same conductance. We recorded from 10 cells with
electrodes containing ADP-ribose (300 ␮M); after 4 min, when the
inward current reached its steady value, we applied a depolarization to
⫹10 mV for an additional 4 min. This depolarization evoked no additional current. In time-matched controls, the depolarization elicited
large inward currents recorded at ⫺80 mV (Fig. 6).
Discussion
We found that sustained depolarization of rat peritoneal macrophages activates a large inward current. The current is carried by
a conductance that is nonselective for cations, and that also has
quite significant permeability to chloride and gluconate ions. The
permeability to chloride ions was surprising, and for this reason we
compared the results directly with those for the permeability of
another cation-selective membrane channel. In the low chloride
solutions that we have used ([Na]i ⫽ 172 mM, [Cl]i ⫽ 148 mM,
[Na]o ⫽ 44 mM, [Cl]o ⫽ 41 mM), the observed reversal potential
for the P2X2/3 receptor corresponds to a very low chloride permeability (PCl/PNa of ⬃0.02). The estimates of permeability to
NMDG, calcium, and gluconate should be considered as less reliable than those for PCl/PNa. This is because in those cases there
are three permeant ions present, and the estimate involves using
the value previously determined for PCl/PNa.
Likewise, the reversal potential observed with extracellular NMDG
indicates that the P2X2/3 receptor is essentially impermeant (PNMDG/
PNa ⬍ 0.03), whereas both the currents presently studied in macrophages have significant permeability to NMDG. From experiments of
the type described, it is difficult to exclude the possibility that the
sustained depolarization is activating more than one set of ionic channels; for example, one permeable to anions and another permeable to
cations. All that can be said in this case is that each has a very similar,
and linear, current-voltage relation and each has a similar sensitivity
to block by lanthanum. We shall return later to discuss the question of
how a sustained depolarization might lead to the activation of such an
ionic conductance(s).
There are many similarities between these permeation properties
and those reported by Holevinsky and Nelson (1) for the current
following phagocytosis of fluorescein-labeled immune complexes
recorded from macrophages derived from human blood monocytes. That was a large current (typically ⬃1 nA), similar in amplitude to the currents that we presently describe; this is in marked
contrast to the very small (⬃50 pA) current elicited by thapsigargin in the same cells (10). The underlying conductance had
PNMDG/PNa of 0.69 in the work of Holevinsky and Nelson (1),
which is close to our present estimate. Furthermore, they also reported that the underlying channels had significant anion permeability, and that glutamate appeared to be more permeable than
chloride. That current was also completely blocked by 1 mM lanthanum, although the effects of lower concentrations were not examined. Holevinsky and Nelson (1) found that a current with similar properties could be activated by xanthine plus xanthine
oxidase, although not by xanthine alone, and they suggested that
the release of free radicals following phagocytosis activated the
underlying conductance. Holevinsky and Nelson (1) did not investigate whether a sustained depolarization could elicit such a current, as we have reported; they did report that, under current clamp,
the current led to a sustained depolarization of the cells.
It has recently become clear that oxidants such as H2O2 can
indeed activate a cation conductance in certain immune cells (12).
In the monocytic cell line U937, as well as Jurkat T lymphocytes
and the eosinophil line EOL1, this can be mimicked by intracellular ␤NAD⫹, but not by ␤NADH (12, 13). This implies that
␤NAD⫹, formed by the action of free radicals on intracellular
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FIGURE 4. Current activated by intracellular ADP-ribose. A, A large inward current progressively develops during the first 50 s of recording with a
pipette containing 300 ␮M ADP-ribose. At the periods indicated, the superfusion solution was changed to NMDG, isotonic calcium, or lanthanum. B,
Current-voltage plots showing the progressive increase in conductance after beginning whole cell recording with an electrode containing ADP-ribose (300
␮M). Voltage ramps were ⫺130 to ⫹50 mV in 500 ms, repeated every 5 s. Times indicated beside selected traces. C, Time course of development of current
with different ADP-ribose concentrations. Control, no ADP-ribose (E, n ⫽ 4); F, 100 nM (n ⫽ 6); 䡺, 1 ␮M (n ⫽ 3); f, 100 ␮M (n ⫽ 6); and ‚, 300
␮M (n ⫽ 6). The currents are normalized to the value at 240 s in each particular cell; these values were 66 ⫾ 25 pA (4), 309 ⫾ 143 pA (6), 356 ⫾ 145
pA (3), 949 ⫾ 390 pA (6), and 1521 ⫾ 432 pA (6) for control, 100 nM, 1 ␮M, 100 ␮M, and 300 ␮M, respectively. The numbers beside each trace are
the time constants of fitted exponentials. Holding potential ⫺80 mV. D, Concentration-response curve for current measured at 240 s as a function of
intracellular ADP-ribose concentration (n ⫽ 3– 6 cells for each point).
1172
IONIC CURRENTS IN MACROPHAGES
FIGURE 6. Occlusion of currents activated by depolarization and by
ADP-ribose. A, Development of the current in 10 macrophages held at ⫺80
mV for 240 s (F, filled bar); recording electrode contained ADP-ribose
(300 ␮M). At 240 s, the holding potential was changed to ⫹10 mV (E,
open bars); the current at ⫺80 mV was determined from voltage command
ramps. Depolarization to ⫹10 mV did not result in the development of any
additional current. B, Time-matched control recordings in which the electrode did not contain ADP-ribose. Depolarization to ⫹10 mV beginning at
240 s resulted in the development of an inward current (recorded at ⫺80
mV) similar to that illustrated in Fig. 1.
␤NADH, mediates the effect. The molecular identity of the underlying channel has been approached in two ways. First, heterologous expression of TRPM2 cDNA results in the appearance of
channels permeable to cations that can be activated by intracellular
␤NAD⫹ (⫽1 mM) (12, 13). Second, treatment of U937 monocytes
with antisense oligonucleotides that reduced the expression of
TRPM2 (measured by Western blotting and immunocytochemistry) also reduced the calcium entry and cell death elicited by H2O2
(12). These experiments strongly suggest that one or more TRPM2
subunits are a key component of the channel expressed by these
immune cell lines that is activated by oxidative damage (12, 13).
In contrast, it has recently been found that H2O2 can activate heterologously expressed TRPM2 channels by a mechanism that appears not to involve changes in ADP-ribose or NAD levels (23).
The homomeric channels formed by expression of TRPM2 subunits are directly activated also by ADP-ribose (13, 15), although
not by several closely related analogs (cyclic ADP-ribose,
␤NADH, ␤NADP⫹, ␤NADPH, ATP, and ADP) (12, 13, 15). The
activation results from binding of the ADP-ribose to a Nudix domain in the C terminus of the protein, because it is prevented by
point mutations of key residues in this motif (12, 15). The concentration of ADP-ribose causing 50% activation of the whole cell
current is ⬃30 –100 ␮M (15). When activated by intracellular diffusion of ADP-ribose during the course of whole cell recordings,
these TRPM2 currents show close to linear I/V relations and others
properties that resemble the current first described by Wilding et
al. (24) in ascidian oocytes.
The current that we now report in rat peritoneal macrophages,
which is activated by ADP-ribose, has some, but not all, of the
same features. These include the effectiveness of ADP-ribose and
␤NAD⫹ (although not a range of other structurally related compounds), the effective concentrations of ADP-ribose (EC50 ⬃30
␮M; Fig. 4D) and ␤NAD⫹ (1 mM), and the block by ATP (5 mM)
(13). We considered that an excess of ADP might inhibit the binding of ADP-ribose to its Nudix domain (25), and consistent with
this we observed that intracellular ADP also blocked the currents
activated by both ADP-ribose and depolarization. However, there
also appear to be important differences between the properties of
heterologously expressed TRPM2 channels and the macrophage
currents. The most notable is the significant permeability to
NMDG, chloride, and gluconate that we observed, whereas substitution of external cations by NMDG almost completely blocks
the current through homomeric TRPM2 channels (13, 15). Furthermore, we observed a relatively high sensitivity to lanthanum (IC50 ⬇
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Current evoked by superoxide anions. A, Superfusion with
xanthine (1 mM) and xanthine oxidase (0.05 U/ml) evoked an inward current that developed over several tens of seconds (n ⫽ 6). This was rapidly
and completely blocked by lanthanum (100 ␮M). After washout of lanthanum, depolarization evoked the usual current in the same cells. B,
Changes in intracellular sodium were measured as SBFI fluorescence. Application of H2O2 induced a slow rise in [Na]i (E) that was strongly reduced by
lanthanum (1 mM; f). Control cells (F, no H2O2 added) showed only a small
rise in [Na]i during the same time period. Values are mean ⫾ SEM for 10 cells.
The Journal of Immunology
Acknowledgments
We thank Daniele Estoppey and Gareth Evans for assistance with cell
culture, and Claire Fenech for advice on sodium imaging.
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1–10 ␮M) on the depolarization-evoked and ADP-ribose-evoked current in peritoneal macrophages, but the sensitivity of TRPM2 currents
to lanthanum has not yet been studied systematically.
In contrast, other currents have been described with properties
that more closely resemble the macrophage currents observed in
the present study. For example, heterologous expression of PKD1
and PKD2, genes named for the polycystic kidney disease that
arises when they are mutated in humans (26), gives rise to a similar
conductance (27). Those channels have PNMDG/PNa of 0.51. The
PCl/PNa cannot be calculated directly from the data of Hanaoka et
al. (27) (they had three permeant ions present: cesium, sodium, and
chloride); however, the 45 mV shift in reversal potential would
indicate PCl/PNa of ⬃0.3. The lanthanum sensitivity of the PKD1/2
current (50% inhibition by ⬃50 ␮M) is also not very different from
that which we observed in peritoneal macrophages. It is not clear
how polycystin channels could be activated by ␤NAD⫹ and ADPribose, because their amino acid sequence does not contain a Nudix domain. Further work, such as recordings of single channel
activity and antisense oligonucleotide approaches, will be needed
to establish the molecular composition of the macrophage channel.
The occlusion experiment illustrated in Fig. 5 strongly suggests
that the two currents that we describe pass through the same channels (although it is also formally possible that intracellular ADPribose could in some way inhibit the effect of depolarization to
activate a separate population of channels). This is consistent with
the overall similarity in the properties of the currents with respect
to permeability (Table I) and lanthanum blockade (Fig. 3). This
raises the question deferred earlier: what might be the mechanism
by which sustained depolarization activates this cell conductance?
Speculations might include that depolarization evokes the release
of ADP-ribose from intracellular stores, or in some way facilitates
its synthesis from ␤NAD⫹. In the latter regard, it is worth noting
that the enzyme responsible for the formation of ADP-ribose from
␤NAD⫹ (CD38, NAD-glycohydrolase) is itself membrane associated, with its enzymatic activity in the ectodomain (28, 29). It is
possible that the movement of negatively charged ADP-ribose into
the cell is in some way facilitated by the sustained applied depolarization. Our observation that the current elicited by depolarization and the current evoked by intracellular ADP-ribose had very
similar sensitivities to blockade by intracellular ATP and ADP
(Fig. 3B) is obviously consistent with the interpretation that ADPribose serves as the messenger for the depolarization. In this case,
as with the experiments using xanthine and xanthine oxidase, it
must be argued the involvement of ADP-ribose is local rather than
distributed throughout the cytoplasm; this is because the responses
were well maintained in cells during relatively long periods of
intracellular dialysis with the contents of the recording pipette.
In conclusion, an imposed depolarization of macrophages activates an inward current that is significantly permeable to large
cations as well as anions, and that is blocked by low ␮M concentrations of lanthanum. Under more physiological circumstances
(i.e., without voltage-clamp), this current could amplify the depolarization by opening further channels. In this way, the effect of an
initial stimulus that leads to the release of free radicals within the
cell (such as phagocytosis) would be further enhanced. Such a
positive feedback process, in which activation of the current leads
to depolarization of the macrophage and depolarization leads to
further activation, might contribute to the eventual cell death that
readily follows exposure to exogenous oxidants.
1173