Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-900 Rio de Janeiro, Brasil
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ABSTRACT |
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Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 ± 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-D-glucamine, and glutamate; their opening is favored at temperatures higher than 30°C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3'-O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.
adenosine 5'-triphosphate; permeabilization; 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate; oxidized adenosine 5'-triphosphate; uridine 5'-triphosphate; P2X7
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INTRODUCTION |
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IT HAS BEEN WELL ESTABLISHED that extracellular ATP (ATPo) may trigger intracellular signaling pathways, open ion channels, and induce different physiological responses depending on cell type and P2 purinoceptors expressed [reviewed by Dubyak and El-Moatassim (12)]. Five types of receptors, termed P2Y, P2U, P2X, P2T, and P2Z, have been identified based on pharmacological and functional studies (7, 15). Recently, based mainly on the analysis of protein sequences and signal transduction mechanisms, a new nomenclature scheme has been proposed for these receptors (5, 15, 36). According to this view, there are two major families of P2 purinoceptors, one with the properties of intrinsic ion channels, termed P2X, and the other coupled to G proteins, termed P2Y.
The term P2Z continues to be used to name receptors associated with the
opening by ATP4 of a
nonselective, poorly characterized ion pore (11, 15). Its presence has
been described in many tissues and systems, including bone
marrow-derived cells such as macrophages, mast cells, thymocytes, some
lymphocytes, the phagocytic cells of the thymic reticulum (PT-R cells),
and Langerhans cells (9, 11, 27, 38, 39). P2Z receptors have been
frequently detected indirectly by the permeabilization of the cell
membrane to fluorescent dyes such as lucifer yellow and ethidium
bromide that happens a few minutes after
ATPo addition (34, 39).
Macrophages and mast cells are permeable to solutes of up to 900 Da,
whereas some lymphocytes, thymocytes, and hematopoietic stem cells are
permeable to solutes of up to 400 Da (12, 15, 27).
The physiological function of P2Z purinoceptors in the immune system is still an open question (11, 12, 29). In macrophages, it has been associated with interleukin-1 maturation and release (21), formation of multinucleated giant cells (14), and elimination of macrophages infected by intracellular parasites (22). However, due to the strong permeabilization phenomenon and the induction of apoptosis in some cell types such as thymocytes and macrophages, a role in cell death has been proposed (11).
Patch-clamp studies have generated valuable information regarding the interaction of ATP with P2Z purinoceptors. However, experiments performed in mast cells (38) and macrophages (1, 6, 18) have so far failed to detect single-channel currents that could be associated with an ATPo-induced pore. In macrophages and PT-R cells, two currents can be promptly induced by ATPo: a depolarizing current that is selective for small monovalent cations and a Ca2+-dependent K+ current (1, 9, 17). We have recently shown that this depolarizing current can be ascribed to a 5- to 8-pS channel that is too small to explain the permeabilization phenomenon (10). Moreover, the proposal of involvement of hemi-gap junction channels formed by connexin-43 (3) could not be confirmed (2).
To further investigate the nature of the P2Z-associated permeabilization phenomenon, we performed experiments using the cell-attached configuration of the patch-clamp technique in macrophages under conditions known to induce permeabilization. These experimental conditions would avoid modifications of the intracellular environment and increase our chances of obtaining direct electrophysiological recordings of a putative P2Z pore. Here, we describe for the first time single-channel currents of large nonselective channels opened by ATPo in mouse peritoneal macrophages and J774 cells. The conductance, selectivity, and pharmacological characteristics of these pores are consistent with the expected properties of a P2Z-associated pore.
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MATERIALS AND METHODS |
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Cells. Thioglycolate-elicited macrophages were obtained from the intraperitoneal cavity of Swiss-Webster mice. Cells were transferred to RPMI 1640 medium containing 5% heat-inactivated fetal calf serum, 2 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin and were plated in 35-mm petri dishes. All surgical manipulations were performed under ether anesthesia. After 1 h of incubation at 37°C in a 5% CO2 humidified atmosphere, nonadherent cells were removed, and the adherent cells were kept in the same conditions for 4 h to 15 days until use. Unless otherwise specified, we used macrophages in our experiments. In some experiments, the mouse macrophage J774 cell line (J774 cells) was used. The cells were grown in 25-ml tissue culture flasks kept at the same conditions as above and were plated in 35-mm petri dishes for 2 h to 4 days before use.
Reagents. ATP, UTP, ADP,
3'-O-(4-benzoylbenzoyl)-ATP
(BzATP), oxidized ATP, ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), tris(hydroxymethyl)aminomethane (Tris),
N-methyl-D-glucamine (NMDG),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), and sodium glutamate were purchased from Sigma
Chemical (St. Louis, MO). The pore-forming protein of cytotoxic
lymphocytes (perforin) was purified from the CTLL-R8 cell line by
low-pressure liquid chromatography using a Q-Sepharose column coupled
to an fast-performance liquid chromatography system
(Pharmacia Biotech, Uppsala, Sweden) as previously described (20, 30).
Perforin activity (5 hemolytic U/µl) was quantified in a
Ca2+-dependent hemolytic assay as
described previously (20).
Electrophysiological measurements.
Ionic currents were studied in cell-attached and, in some cases, whole
cell configuration, using an EPC-7 amplifier (List Electronic,
Darmstadt, Germany) according to standard patch-clamping techniques
(16). The petri dishes containing the cells were filled with 10 ml of
solution and were placed in the heated stage of a microscope. Gigaohm
seals were formed after offset potential compensation, using
heat-polished micropipettes of 5-10 M. ATP, UTP, and ADP (100 mM) were applied to 10 ml of extracellular solution by manually
dropping 50-100 µl of solution into the plate dish. BzATP (100 µl, 14 mM) was applied to 7 ml of extracellular solution. All drugs
were diluted in normal extracellular solution and were stored in the
dark at
20°C until use. Temperature was continuously
monitored with a digital thermometer placed in the extracellular
solution and, unless otherwise specified, was maintained in the range
of 30-37°C.
Current and voltage were simultaneously recorded on a paper chart recorder (Mark VII WR 3310; Graphtec, Yokohama, Japan) and in a VCR tape, and digitalization was performed by a Neurocorder (model DR-390; Neuro Data Instruments) for off-line analysis. Conductances of unitary channels were obtained by manually fitting a straight line throughout the current value corresponding to the open state of neighbor events and then measuring the amplitude. Current-voltage (I-V) curves were then plotted, and linear regressions were performed using the SigmaPlot software (Jandel). Figures 1-6 are representative records of each type of experiment described in the text. I-V plots in Figs. 1-3 were taken from single channels of the same patches. Mean values and the number of experiments (n) are described in the text and in Table 1. Current signals were filtered at 3 kHz during acquisition and at 300 Hz during the play back of the recordings. To correct for junction potentials, the ground electrode was placed in a chamber containing the same solution as the patch pipette and was connected to the extracellular solution by an agar bridge. The diffusion potentials at the tip of the patch pipette were then measured for each intrapipette solution at the beginning of each experiment, as described by Neher (26). All voltages shown in Figs. 1-6 refer to the holding potentials (VH) inside the patch pipette, without any corrections, whereas the numeric values of the reversal potentials (VRev) shown in the text and in Table 1 are corrected for the pipette junction potential. Average data are given as means ± SD.
Unless otherwise specified, the compositions of the extracellular and
intrapipette solutions were the same (in mM): 150 NaCl, 5 KCl, 1 MgCl2, and 10 Na-HEPES, pH 7.4 (normal extracellular solution). In the whole cell experiments, the
intrapipette solution contained (in mM) 150 KCl, 5 NaCl, 1 MgCl2, 0.1 K2-EGTA, and 10 K-HEPES, pH 7.2 (normal intracellular solution). Ion substitution experiments were
performed by using the following intrapipette solutions: in
low-Na+ solutions, 150 mM of
either Tris · Cl or NMDG-Cl substituted for NaCl; in
low-Cl solution, 150 mM
sodium glutamate substituted for NaCl. Low NaCl contained (in mM) 34 NaCl, 158 mannitol, 0.12 CaCl2,
0.5 MgCl2, 1.4 EGTA, and 5 HEPES,
pH 7.4. Typical junction potentials were 4,
5.5, and
3.5
mV for low-Na+,
low-Cl
, and low-NaCl
solutions, respectively. Normal extracellular solution did not require
any correction.
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RESULTS |
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Single-channel currents induced by ATPo
in cell-attached patches.
Because previous patch-clamp experiments have failed to detect
P2Z-associated pores (1, 18, 24, 25), we decided to investigate the
occurrence of any ATPo-triggered
ion channel in cell-attached patches at 30-37°C using normal
extracellular solution both in the patch pipette and in the
extracellular bath. These conditions avoided any disturbance to the
intracellular medium and assured that most macrophages would become
permeable to lucifer yellow upon ATP addition. Moreover, because the
transduction signals involved in the permeabilization phenomenon may
require second messengers (4), ATP was added to the extracellular
solution only after gigaseal formation. As shown in Fig.
1A,
addition of normal extracellular solution in the proximity of the cell
did not induce any ion channel activity on the patch, whereas addition of ATP induced the opening of several ion channels. At pipette VH ranging from
20 to
60 mV, larger steps of current were frequently observed. To evaluate channel conductances and
VRev, the resting transmembrane potential of the cell was measured in independent current-clamp whole cell experiments performed at least 30 s after addition of ATPo. Using normal
intracellular solution in the pipette, we obtained a value of
1 ± 2 mV (n = 6), consistent with
the already described depolarization induced by
ATPo (1, 6, 18).
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Selectivity of ATPo-induced channels.
The size and the
VRev of the
channels described in Fig. 1 suggested that they could be
nonselective, allowing the passage of both cations and anions of
different sizes, as it would be expected for a pore involved in the
P2Z-associated permeabilization phenomenon. To investigate this
question, we have performed cell-attached ion substitution experiments
using four intrapipette solutions containing different concentrations
of cations and anions. Junction potentials were taken into account as
described in MATERIALS AND METHODS. In all solutions, we
observed large channels displaying VRev close to 0 mV, but, in some conditions, the channels had at least two different
sizes (Figs. 2 and
3 and Table 1). When NMDG
[relative molecular weight
(Mr) of
195] substituted for Na+
(Fig. 2A), a channel of 350 ± 30 pS (n = 8) and
VRev of
2.3 ± 2.5 mV were observed. When Tris
(Mr 121)
substituted for Na+ (Fig. 2,
B, C,
and E), channels with two different
conductances (340 ± 20 pS, n = 3 and 221 ± 31 pS, n = 6)
were observed. Both had
VRev close to 0 mV and could be clearly distinguished in
I-V plots (Fig. 2E and Table 1).
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Other properties consistent with a P2Z-associated pore.
To further characterize the above-described pore(s) as involved in the
P2Z-associated permeabilization, we investigated other known properties
of this phenomenon (7, 11, 12, 23, 34): temperature dependence,
triggering by BzATP, lack of effect of ADP and UTP, and blockade by
Mg2+ and oxidized ATP (Fig.
4). In this series of experiments, we kept
the intrapipette
VH in the range
from 20 to
30 mV since, under this condition at 37°C,
ATPo induces pore opening in <30 s, as shown in Fig. 1.
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DISCUSSION |
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The P2Z purinoceptor-associated permeabilization phenomenon was described in macrophages and mast cells more than 10 years ago (8, 35, 38). However, all whole cell and single-channel currents described so far in these cells do not have the characteristics expected for a cation and anion nonselective pore. A protein called P2X7 has been recently cloned and displayed the pharmacological and functional characteristics of P2Z receptors when expressed in several cell lines (37). ATPo-induced membrane permeabilization and a cation current similar to the ones present in macrophages were observed in these cells, but no single channels of pores were recorded from P2X7-transfected cells (37).
In the search of possible reasons for the lack of electrophysiological
data corresponding to an
ATPo-induced pore, we performed cell-attached patch-clamp experiments in macrophages submitted to
conditions known to induce permeabilization. Here, we describe the
presence of large nonselective channels in macrophages and J774 cells.
The finding that these channels are permeable to large cations and
anions such as Tris, NMDG, and glutamate
(Mr 121-195 Da) suggests that they are involved with the phenomenon of
P2Z-associated permeabilization. Therefore, we named them Z-1 and Z-2
pores, for the larger and the smaller pores, respectively. In
accordance with this conclusion is the fact that these
ATPo-induced pores display several
other properties of the P2Z-associated permeabilization (7, 11, 12, 34;
see Fig. 4); they are temperature dependent and are not triggered by
ATPo in the presence of high
extracellular Mg2+ concentration,
indicating the requirement for
ATP4. Moreover, BzATP, but
neither ADP nor UTP, can substitute for ATP in the induction of pore
activity. In addition, oxidized ATP, an inhibitor of the
permeabilization phenomenon, also inhibits pore activity. Taken
together, our results lead to the conclusion that Z pores are involved
in the P2Z-associated permeabilization phenomenon. However, more
experiments are needed to establish the size limit of the molecules
that can diffuse through these pores and compare it with the know
values of the permeabilization phenomenon.
In several membrane patches, other channels could be observed under conditions in which Z pores were not opened (e.g., Fig. 4G). This observation could be explained by the activation of ATP-induced channels via other P2 receptors present in the macrophage membrane. Two possibilities are the activation of Ca2+-dependent K+ channels (1, 17) and the ATP-activated Ca2+-permeable channel described by Naumov et al. (24, 25). In this regard, it should be noticed that oxidized ATP does not inhibit the ATP-induced increase of the intracellular Ca2+ concentration (23).
Only Z-1 pores were clearly present when normal extracellular solution
was used in the patch pipette (Fig. 1 and Table 1). Its conductance
(409 pS) is in accordance with the expected value of a pore permeable
to lucifer yellow
(Mr of 457 and
Stokes radius of 7.8 A; see Refs. 32, 38). Z-2 pores were more evident
in solutions with low Na+
and/or low Cl
concentration. However, although data were not always enough to plot
I-V
curves, single-channel activity or steps of current compatible with Z-2
pores were also observed under all conditions studied here (data not
shown). It is not clear at the moment whether Z-2 represents an
independent channel or a subconductance state of the Z-1 pore. However,
the existence of two P2Z-associated pores or opening states is
consistent with data showing that, although macrophages are permeable
to molecules of up to 900, some lymphocytes seem to have a molecular
weight cut-off of ~400.
The observation that the Z pores opened in membrane patches isolated from the ATP-containing extracellular medium by the gigaohm seals indicates that these pores are coupled to purinoceptors by a pathway involving second messengers. This conclusion is consistent with previous experiments that failed to obtain large conductance steps in whole cell experiments in which the intracellular milieu was not preserved (1, 18). We have not elucidated the nature of the second messengers involved in the opening of Z pores. However, it is interesting to note that recent evidence suggests the involvement of calmodulin and phospholipase D in the permeabilization phenomenon (4, 19, 28).
On the other hand, it has been demonstrated that the P2Z/P2X7 receptor is a ligand-gated channel associated with a cation current that displays a single channel conductance of 5-8 pS, not directly involved in the transport of low-molecular-weight solutes (1, 10, 12, 37). These results can be conciliated with the second-messenger hypothesis by proposing that, although Z pores are activated by the P2Z/P2X7 receptor, they are distinct channel proteins. Alternatively, Z pores could be a new (second messenger-dependent) activation state of the P2Z/P2X7 receptor itself.
There is indeed some evidence in the literature indicating that permeabilization can be separated from other P2Z-associated phenomenon: differential activation of the cation current and the nonselective pores can be achieved in Xenopus oocytes expressing macrophage mRNA (28); calmodulin antagonists are able to prevent the lytic effects of ATPo without affecting calcium influx and membrane depolarization (4); and, in P2X7-transfected cells, permeabilization, but not the cation current, is dependent on the cytoplasmatic tail of the P2X7 protein (37). The full elucidation of this problem will require the identification of the intracellular pathways triggered by P2Z receptors and the cloning of the permeabilization pore(s).
One interesting property of Z-1 and Z-2 pores is that they tend to be closed at negative transmembrane potential (positive VH). This finding suggests that, in macrophages, they can be regulated by the balance of two opposing mechanisms also induced by ATPo: the fast depolarization caused by the small cation channels recently described by us (10) and the delayed and transitory Ca2+-dependent K+ current that follows the first one (1, 17, 29). These same mechanisms would also regulate the P2Z-associated permeabilization phenomenon. In this regard, it is interesting to notice that high extracellular K+ concentration, a condition that depolarizes the cells, enhances permeabilization in peripheral blood lymphocytes (Ref. 39 and our unpublished observations).
The cascade of steps shown in Fig. 6 is possibly associated with Z-1 pores and permeabilization. However, more data are needed to clearly establish this connection. The explosive nature of the events suggests a cooperative phenomenon in which the opening of a first pore facilitates the opening of the next ones. This pattern of conductance increase has already been described for the insertion of pores of perforin in cell membranes (31).
Our results suggest that the Z pores described in this report are triggered by P2Z purinoceptors and are involved in the permeabilization of the macrophage plasma membrane to low-molecular-weight solutes. The study of these pores may contribute to the understanding of the mechanism and functional role of plasma membrane permeabilization induced by ATPo in macrophages and other cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Vandir da Costa for continuous technical support, to Andreia Lamoglia de Souza for helping to purify perforin, and to Drs. Luiz A. Alves and Masako O. Masuda for critical reviews of the manuscript and helpful discussions.
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FOOTNOTES |
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This work was financed by grants from Conselho Nacional de Desenvolvimento Cientifico e Technológico do Brasil, Financiadora de Estudos e Projetos, and Fundaçao de Amparo à Pesquisa do Estado do Rio de Janeiro.
Address for reprint requests: P. M. Persechini, Laboratório de Imunobiofísica, Instituto de Biofísica Carlos Chagas Filho da UFRJ, Bloco G do CCS; Ilha do Fundão, 21941-900 Rio de Janeiro, Brazil.
Received 9 January 1997; accepted in final form 8 August 1997.
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