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Previous Article | Next Article
The Journal of Neuroscience, August 15, 2000, 20(16):5965-5972
P2X7 Receptors in Müller Glial Cells from the Human Retina
Thomas Pannicke1, Wolfgang Fischer2, Bernd Biedermann1, Hiltrud Schädlich2, Jens Grosche1, Frank Faude3, Peter Wiedemann3, Clemens Allgaier2, Peter Illes2, Geoffrey Burnstock4, and Andreas Reichenbach1 1 Paul-Flechsig-Institute for Brain Research, University of Leipzig, 04109 Leipzig, Germany, 2 Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04103 Leipzig, Germany, 3 Department of Ophthalmology, University of Leipzig, 04103 Leipzig, Germany, and 4 Autonomic Neuroscience Institute, Rowland Hill Street, London NW3 2PF, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
ATP has been shown to be an important extracellular signaling molecule. There are two subgroups of receptors for ATP (and other purines and pyrimidines): the ionotropic P2X and the G-protein-coupled P2Y receptors. Different subtypes of these receptors have been identified by molecular biology, but little is known about their functional properties in the nervous system. Here we present data for the existence of P2 receptors in Müller (glial) cells of the human retina. The cells were studied by immunocytochemistry, electrophysiology, Ca2+-microfluorimetry, and molecular biology. They displayed both P2Y and P2X receptors. Freshly enzymatically isolated cells were used throughout the study. Although the [Ca2+]i response to ATP was dominated by release from intracellular stores, there is multiple evidence that the ATP-induced membrane currents were caused by an activation of P2X7 receptors. Immunocytochemistry and single-cell RT-PCR revealed the expression of P2X7 receptors by Müller cells. In patch-clamp studies, we found that (1) benzoyl-benzoyl ATP (BzATP) was the most effective agonist to evoke large inward currents and (2) the currents were abolished by P2X antagonists; however, (3) long-lasting application of BzATP did not cause an opening of large pores in addition to the cationic channels. By microfluorimetry it was shown that the P2X receptors mediated a Ca2+ influx that contributed a small component to the total [Ca2+]i response. Activation of P2X receptors may modulate the uptake of neurotransmitters from the extracellular space by Müller cells in the retina.
Key words: Müller cells; glia; P2 receptors; ATP; glutamate uptake; human
INTRODUCTION
Extracellular purines have been described as transmitter molecules in various cells in different tissues. Like various other ligands, ATP is thought to activate several distinct receptor types. The current nomenclature includes the G-protein-coupled P2Y family of receptors and the P2X receptors that are ligand-gated ion channels (Burnstock, 1997
). ATP-activated ion channels have been found in neurons and muscle cells (Bean, 1992
MATERIALS AND METHODS
Materials. The following drugs were used: ATP (Serva, Heidelberg, Germany), adenosine 5'-O-(2-thiodiphosphate) trilithium (ADP
S), oxidized ATP, 2'- and 3'-O-(4-benzoyl-benzoyl)-ATP triethylammonium (BzATP), fura-2 AM, Lucifer yellow dilithium,
,
-methylene-ATP disodium (
Other substances were from Sigma unless indicated otherwise.
Preparation of cells. Human retinal tissue was obtained during vitreoretinal surgery in cases of proliferative vitreoretinopathy when retinectomies were indicated, as well as from organ donors whose eyes served as a source for corneal transplantation. Use of human retinae was approved by the ethics committee of the School of Medicine, University of Leipzig.
The procedure for the preparation of Müller cells has been described previously (Reichenbach and Birkenmeyer, 1984
For dye-filling experiments, the cells were incubated at room temperature (20-24°C) or 37°C in PBS (Ca2+/Mg2+-free) containing the fluorescent dye and BzATP (50 and 100 µM). Under control conditions, cells were kept without BzATP. After the incubation period, cells were investigated using epifluorescent equipment (for all of the fluorescent dyes that were used: excitation bandpass 450-490 nm, emission long-pass 520 nm; additionally for ethidium bromide: excitation 546 nm, emission long-pass 590 nm; Zeiss, Germany). A part of the experiments using the dye Alexa Fluor 488 was performed at a laser scanning microscope (LSM 510, Zeiss, Germany) with an excitation wavelength of 488 nm and a long-pass for emission at 505 nm.
Preparation of antibodies and immunocytochemistry. Specific polyclonal antibodies were prepared to the C-terminal part of the human and rat P2X7 receptor as described previously (Oglesby et al., 1999
Preparations of dissociated cells were used for immunocytochemical demonstration of P2X7 receptors. Isolated Müller cells were fixed in 4% paraformaldehyde (20 min), washed in PBS, and air-dried. For blocking and permeabilization, the cells were pretreated in PBS containing 5% normal goat serum (Dianova, Hamburg, Germany), 1% DMSO, and 0.3% Triton X-100 for 1 hr. Afterward, the samples were incubated 12 hr at 4°C with the primary antibodies, diluted 1:400. After washing, the slides were incubated with Cy3-tagged goat anti-rabbit IgG (Dianova) at a 1:500 dilution for 1 hr at room temperature. After a wash step, the specimens were dehydrated and mounted in Entellan (Merck, Darmstadt, Germany). The results were documented by using the LSM 510.
Electrophysiology. For patch-clamp recordings, the cells were suspended in extracellular solution in a recording chamber mounted on the stage of an upright microscope (Axioskop, Zeiss, Germany). Control extracellular solution contained (in mM): NaCl 110, KCl 3, CaCl2 2, MgCl2 1, Na2HPO4 1, glucose 11, HEPES-Tris 10, NaHCO3 25, and was equilibrated to pH 7.4 by continuous bubbling with carbogen gas (95% O2, 5% CO2). If not otherwise indicated, responses to ATP and its analogs were recorded in a solution where Ca2+, Mg2+, and K+ ions were replaced by Na+. Removal of K+ was performed to suppress the dominating K+ conductance of the Müller cells. The divalent cations would reduce the concentration of ATP4
(or the corresponding forms of its analogs), which is suggested to be the species active at the P2Z (=P2X7) receptor (Di Virgilio, 1995
Recording electrodes were made from borosilicate glass (Science Products, Hofheim, Germany) and had resistances of 4-6 M
if filled with an intracellular solution containing (in mM): NaCl 10, CsCl 130, CaCl2 1, MgCl2 2, EGTA 10, HEPES-Tris 10, pH 7.1. In some experiments with K+ in the extracellular solution, CsCl was replaced by KCl. After a seal was established, the recording chamber was continuously perfused (2 ml/min) with extracellular solution. All agonists and antagonists were applied by the bath perfusion. Experiments were performed at room temperature (20-24°C).
Recordings in the whole-cell configuration of the patch-clamp technique were performed using the patch-clamp amplifier Axopatch 200A (Axon Instruments, Foster City, CA). Currents were low-pass-filtered at 1 kHz with an eight-pole Bessel filter and digitized at 5 kHz using a 12 bit-A/D converter. Voltage command protocols were generated and data analysis was performed with the software ISO 2 (MFK, Niedernhausen, Germany). Unless indicated otherwise, data are given as mean values with SD. For curve fitting the software SigmaPlot (Jandel Scientific, Corte Madera, CA) was used.
Ca2+-microfluorimetry. Isolated Müller cells in MEM (see above) were seeded in the center of glass coverslips coated with poly-L-lysine. After attachment of the cells (15 min), the preparations were loaded with the Ca2+-sensitive fluorescent dye fura-2 AM (5 µM) for 30 min and then incubated in Mg2+-free physiological saline containing (in mM: NaCl 135, KCl 5, CaCl2 0.5, HEPES 10, D-glucose 10, pH 7.2, adjusted with Tris base) for an additional 20-30 min to remove traces of extracellular fura-2 AM and to complete de-esterification. After that, the coverslips were mounted cell-side up into the free bottom of a perfusion chamber (250 µl) placed on the stage of an inverted microscope with epifluorescence optics (Diaphot 200, Nikon, Japan). Throughout the experiments, cells were continuously superfused (at 0.8 ml/min) by means of a roller pump with control and drug-containing solutions, respectively. Different superfusion solutions were selected with a valve bank coupled to several reservoirs. ATP and various P2 receptor agonists were applied for 30 sec (intervals between two applications 10-12 min); Ca2+-free solution (always with 1 mM EGTA) and modulating drugs such as CPA, thapsigargin, or KN-62, were superfused 10 min before and during additional application of the ATP derivatives. Fluorescence ratio measurements were made on selected single Müller cells with a dual wavelength spectrometer. Fura-2 AM fluorescence (over the cell soma), excited alternatively at 340 and 380 nm, was measured at 510/520 nm by a microscope photometer attached to a photomultiplier detection system (Ratiomaster System, Photon Technology International, Wedel, Germany). Data are expressed as ratios of the fluorescence intensities at 340 versus 380 nm. Complete data acquisition, presentation, and analysis were performed computer-controlled, using the software FeliX, Vers. 1.1 (Photon Technology International). All experiments were performed at room temperature in the dark. Background fluorescence was minimal and was thus not corrected.
Molecular biology. Subsequent to electrophysiological characterization of individual Müller cells by measuring the response to BzATP, single-cell RT-PCR was performed. Cytoplasm from the endfoot region was harvested into the patch pipette containing 6 µl intracellular solution (with KCl) by applying negative pressure. Only BzATP-responsive cells with the seal remaining stable during suction were used for further steps. The content of the patch pipette was expelled into a PCR-tube containing 2 µl dNTP/N6 mix (2.5 mM/25 µM), 1 µl dithiothreitol (100 mM), 0.5 µl RNase inhibitor (20 U; Stratagene), and 1 µl 5× first-strand buffer. Subsequently, Superscript-II-reverse transcriptase (0.5 µl; Life Technologies) was added, and after an additional 5 min at room temperature the reverse transcription was performed at 45°C for 1 hr. Controls were performed by advancing the patch pipette into the bath solution and using its content for RT-PCR; in no case were false amplifications obtained.
Amplification of the P2X7-specific cDNA fragment was performed by nested hot-start PCR with primers selected by OLIGO 5.0 (MedProbe, Oslo, Norway). The first PCR amplification was performed using the primer pair P2X7 sense (outer) (5'-ATCGTGGAGAATGGAGTGAAG-3', binding position 249) and P2X7 antisense (5'-GGATGGCAGTGATGGAAC-3'; binding position 840) yielding a 591 bp fragment. PCR was performed on a PTC-200 thermal cycler (MJ Research) under hot-start conditions using 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer, Norwalk, CT) and each primer in a final concentration of 100 nM. Reaction conditions were as follows: initial denaturation (94°C for 2 min), 40 cycles of denaturation (94°C for 30 sec), annealing (62°C for 30 sec), and a final extension at 72°C for 10 min. In the second PCR amplification, only the sense primer was replaced by P2X7 inner (5'-TGTAAAAAGGGATGGATGGAC-3'; binding position 429). Five microliters of each reaction were amplified for an additional 45 cycles at an annealing temperature of 60°C. The 411 bp amplification product was analyzed by ethidium bromide staining subsequent to agarose gel electrophoresis (1.5%). To verify the identity of amplified DNA, the PCR product was sequenced using BigDye Terminator chemistry (PE Applied Biosystems, Foster City, CA). The sequence was analyzed on a DNA sequencer ABI 377 (PE Applied Biosystems, Foster City, CA).
RESULTS
Immunocytochemistry
Dissociated Müller cells were subjected to anti-P2X7 receptor immunocytochemistry (Fig. 1). Although no specific signal was found in negative controls (omission of the primary antibody) (Fig. 1A), prominent immunoreactivity was found in the somata of all Müller cells (Fig. 1B, arrow); it should be noted that there was considerable autofluorescence in substructures of the photoreceptor cells that often adhered to the Müller cells (Fig. 1A, empty arrow). The dominant somatic (nuclear) anti-human P2X7 receptor label in the Müller cells was accompanied by a rather weak immunoreactivity in their cytoplasmic membranes. An antibody was available against a C-terminal amino acid sequence of the rat P2X7 receptor differing by only one amino acid from that of the human receptor. Thus, we also applied the antibody directed against the rat receptor (Fig. 1C). This antibody revealed specific labeling in dissociated Müller cells. In comparison to the anti-human P2X7 receptor immunoreactivity, the somatic/nuclear label was less strong, but distinct punctate label was observed along the cytoplasmic membranes (Fig. 1C, arrowheads). The endfoot of the cell is shown at a higher magnification in Figure 1C (inset). The label was uniformly distributed along the entire cell membrane.
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Figure 1. Immunocytochemistry for P2X7 receptors in enzymatically dissociated Müller cells. Vitread endfeet are located at the top of the photographs, and distal processes with adhering photoreceptors are orientated downward. A negative control was performed by omission of the primary antibody. Autofluorescence of photoreceptor cells is clearly visible (empty arrow). B, C, Immunolabeled cells with a primary antibody against human P2X7 receptor (B) and against rat P2X7 receptor (C). Strong labeling in the somatic region was found with the human-specific antibody (B, arrow), whereas use of the rat-specific antibody resulted in a more uniform label distributed over the whole cell (C, arrowheads). The inset in C shows the endfoot of the cell at a higher magnification to demonstrate the punctate labeling pattern.
BzATP-evoked inward currents and depolarizations in human Müller cells
When ATP was applied to isolated human Müller cells, small inward currents were evoked. The current amplitude was 63 ± 43 pA (n = 13) with 1 mM ATP (see Fig. 4C). Significantly larger currents were evoked by application of the P2X7 agonist BzATP. When applied at various concentrations, BzATP evoked inward currents in all Müller cells investigated (n = 271).
The amplitude of these inward currents increased in a concentration-dependent manner up to 1 mM BzATP (Fig. 2A) (K+-free conditions). A concentration of 5 µM BzATP was necessary to evoke a measurable inward current, whereas application of 1 µM BzATP resulted in an increase in the noise (n = 3), or in very small inward currents (n = 3), or had no effect (n = 3). The mean amplitude was 190 ± 181 pA with 50 µM BzATP (n = 120). For individual cells, this value ranged between 20 and 896 pA.
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Figure 2. Dose and voltage dependence of BzATP-evoked currents. A, Dose-response curve of BzATP in human Müller cells. For each cell, the inward current after application of 50 µM BzATP was set as 100% (absolute value: 405 ± 242 pA, n = 15); inward currents at the other concentrations are given relative to this value. Data are mean values with SDs; for each concentration at least six cells were recorded. A fit of the data to the Hill equation resulted in an EC50 of 33.9 µM. The inset shows original current responses from one cell to the indicated concentrations of BzATP. Recordings were performed at a holding potential of 80 mV. B-E, Current-voltage (I-V) relationships of BzATP-evoked currents. Whole-cell currents were recorded at different holding potentials both under control conditions (
) and after application of 50 µM BzATP (
). The difference between the currents under BzATP and under control conditions (
) is given in relative values to normalize the different amplitudes. For normalization, the difference current at a holding potential of
Recordings of BzATP-induced currents at different holding potentials revealed a reversal potential of approximately +10 mV (n = 4) (Fig. 2B,C). The same reversal potential was obtained when K+ was present in both the pipette (130 mM) and the extracellular solution (3 mM; n = 7) (Fig. 2D,E). BzATP-induced currents did not inactivate and were stable during application times of several minutes (n = 8) (Fig. 3A). Moreover, repeated application of BzATP for ~10 sec with intervals of 0.5-1 min evoked currents of constant amplitudes (n = 4; data not shown).
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Figure 3. Effects of prolonged application of BzATP. A, Application of 20 µM BzATP causes an inward current with an almost constant amplitude over 5 min application time; the recovery was complete. The holding potential was
The effect of BzATP application on the resting membrane potential of Müller cells was investigated under current-clamp conditions. Because under K+-free conditions in both intracellular and extracellular solutions the membrane potential was close to the reversal potential of the BzATP-evoked current, the cells depolarized by only 5 ± 1 mV (n = 5). In contrast, in the presence of K+ the cells depolarized by 27 ± 12 mV (n = 8; the resting membrane potential for these cells was
To examine which species of ions were carrying these currents, we replaced extracellular Na+ ions with N-methyl-D-glucamine and choline. This prevented the inward current at negative holding potentials (Fig. 3C), whereas outward currents could be evoked by BzATP at positive holding potentials (n = 9; data not shown). It is concluded that the currents are carried mainly by Na+ (inward) and Cs+ or K+ (outward) ions.
The effects of divalent cations on the BzATP-evoked current were examined by applying extracellular Ca2+ and Mg2+ in concentrations from nominally 0 up to 2 and 1 mM, respectively, i.e., in concentrations corresponding to or below physiological levels (Fig. 4A,B). Both ion species decreased the currents at low concentrations and were able to prevent the flux of currents almost completely at physiological concentrations.
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Figure 4. Pharmacological effects. A, B BzATP was applied in a concentration of 25 µM in the presence of different concentrations of Ca2+ (A) and Mg2+ (B). The number of cells examined for each concentration was between 4 and 10. The inward current recorded in the absence of the divalent cations served as the reference value (100% in each cell). Dose-effect curves were analyzed by fitting the data to the Hill equation. The IC50 was 70 µM for Mg2+ and 84 µM for Ca2+. C, BzATP (50 µM), ATP (1 mM and 100 µM),
Pharmacology
There are some P2 receptor agonists that exhibit a relative specificity for certain receptor subtypes. We examined
Pharmacological studies of P2X receptors have been limited by the lack of highly specific antagonists. Suramin, a blocker of P2 receptors (but also of other receptor types), markedly reduced the BzATP-evoked inward currents. At a concentration of 100 µM, suramin reduced the effect of 50 µM BzATP to 15 ± 24% (n = 5) of the control, whereas 200 µM suramin blocked the effect of 25 µM BzATP completely but reversibly (n = 3) (Fig. 4D). A second antagonist, PPADS, thought to be more selective for P2X than for P2Y receptors, was tested at concentrations up to 100 µM. PPADS at 100 µM blocked the effect of 50 µM BzATP completely in one cell (Fig. 4E), whereas in four other cells and with lower concentrations of PPADS (20, 50, and 75 µM; n = 18) the current was reduced to <40%. The currents showed hardly any recovery after several minutes of washout of PPADS. Moreover, oxidized ATP, an inhibitor of the P2Z receptor in mouse macrophages (Murgia et al., 1993
Effects of BzATP application on glial glutamate transporter currents
To determine whether the P2X receptors on human Müller cells are able to modify the glial transmitter recycling mechanisms, we recorded currents mediated by the glutamate transporter in the absence and presence of BzATP. For these experiments the pipette solution contained 130 mM K+, and K+ (3 mM) was also present in the extracellular solution because the glutamate transporter of Müller cells depends on intracellular K+ (Barbour et al., 1988
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Figure 5. Effect of the P2X receptor on the glutamate uptake current. Glutamate (100 µM) was applied as indicated onto a Müller cell clamped at
Ca2+ microfluorimetry
The existence of receptors for ATP was also demonstrated by fluorescence ratio measurements of intracellular Ca2+ concentrations ([Ca2+]i) using single-cell fura-2 AM microfluorimetry. Repeated application of ATP or the P2X7 receptor agonist BzATP for 30 sec at intervals of 10-12 min evoked Ca2+ signals of consistent amplitudes. However, there was a considerable cell-to-cell variability in the magnitude of the responses. The increase in [Ca2+]i was transient in most cells after ATP application, whereas it was more sustained, or even double-peaked, after BzATP (Fig. 6A). In contrast to what had been observed for the transmembrane currents, 10 µM ATP was more effective than 100 µM BzATP in inducing increases in [Ca2+]i (Fig. 6A). The respective mean peak increases of
fluorescence ratio were 1.67 ± 0.42 and 1.22 ± 0.17 (direct comparison in six cells, p < 0.05, paired t test). It is noteworthy that at lower concentrations of BzATP (10 µM) some cells did not respond at all, whereas others revealed a small but clear [Ca2+]i increase that was sensitive to 1 µM KN-62 (reduction of mean peak amplitude to 32.6 ± 10.5%, n = 3). The P2Y1 agonist ADP
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Figure 6. Intracellular Ca2+ responses elicited by ATP and various P2 receptor agonists in dissociated human Müller cells, based on fluorescence ratio measurements using fura-2 AM microfluorimetry. Agonists were applied with the superfusion flow for 30 sec, as indicated by the bars. A, ATP (10 µM) evoked fast and transient [Ca2+]i rises in most Müller cells tested, whereas BzATP (100 µM) showed more sustained responses, sometimes with a second increase. B, The P2Y1 receptor agonist ADP
In a second series of experiments, the effects of Ca2+-free solution (with 1 mM EGTA, superfusion of the cells for 10 min) was examined on ATP- and BzATP-induced increases in [Ca2+]i. Surprisingly, both the ATP (10 µM) and the BzATP responses (100 µM) (Fig. 6C) were only weakly diminished by removal of extracellular Ca2+ (reduction of mean peak amplitudes to 80.6 ± 8.3%, n = 5, and 78.4 ± 11.4%, n = 6, respectively; p < 0.05 compared with the response in control solution). On the other hand, after superfusion (10 min) with 400 nM thapsigargin (data not shown) or 5 µM CPA (Fig. 6D), two specific inhibitors of the endoplasmatic Ca2+-ATPase producing a depletion of intracellular Ca2+ stores, the response to 100 µM BzATP was markedly suppressed but still clearly detectable. Under these conditions, the signals displayed a long latency and increased only slowly (reduction of mean peak amplitude after CPA to 28.8 ± 2.0%, n = 6; p < 0.01). Finally, the combined superfusion of Ca2+-free solution with additional 5 µM CPA antagonized very efficiently the Ca2+ response elicited by 100 µM BzATP (Fig. 6E) (reduction of mean peak amplitudes to 7.9 ± 1.5% vs control before drug application, n = 3).
It should be noted that these experiments are complicated by the fact that although Ca2+ reduces the BzATP-evoked inward current (Fig. 4), it must be present in the extracellular solution if a Ca2+ influx is to be measured by microfluorimetry. Thus, the relatively small [Ca2+]i responses are not easily comparable to the electrophysiologically recorded inward currents, which are mainly caused by the influx of Na+.
Dye filling
There are several studies demonstrating that the activation of P2Z or P2X7 receptors results in the opening of a nonselective membrane pore permeable to molecules up to 900 Da (Steinberg et al., 1987
Although Rassendren et al. (1997)
In summarizing these results we conclude that application of BzATP does not cause any marked opening of membrane pores large enough to allow fluorescent dyes to enter the cells faster than under control conditions.
Expression of P2X7 receptor mRNA
A total of seven Müller cells were investigated for expression of mRNA encoding P2X7 receptors with single-cell RT-PCR using P2X7-specific primers. Before harvesting cytoplasm, inward currents evoked by application of BzATP were recorded in the whole-cell configuration. In three of these cells, a 411 bp amplification product was detected by gel electrophoresis (Fig. 7). Homology with the known P2X7 gene sequence was verified by sequence analysis.
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Figure 7. Expression of P2X7 receptor mRNA revealed by single-cell RT-PCR. The PCR product with 411 bp length was found in three individual Müller cells in an ethidium bromide-stained 1.5% agarose gel after electrophoresis. The negative control was performed as described in Materials and Methods.
DISCUSSION
P2 receptors in Müller cells
Here we provide evidence that human Müller cells express P2 receptors. This finding is in accordance with reports on amphibian (Keirstead and Miller, 1997
Several subtypes of P2X receptors might be active in Müller cells (Liu and Wakakura, 1998
P2X7 receptors: pharmacological characterization
We show that human Müller cells possess a functional P2X-type receptor for extracellular ATP and its analog BzATP. Several lines of evidence indicate that this receptor corresponds to the human P2X7 receptor. First, specific antibodies directed to the human P2X7 receptor revealed strong and specific immunoreactivity. Second, the P2X7 receptor mRNA was detected in Müller cells by RT-PCR.
Third, a higher affinity for BzATP than for ATP (and other analogs) is thought to be characteristic for the P2X7 receptor (Burnstock, 1997
Fourth, the currents evoked by BzATP were blocked by relatively high concentrations of suramin and PPADS. This is in accordance with data of Rassendren at al. (1997)
The current-decreasing effects of Mg2+ and Ca2+ are well known and were demonstrated for the P2X7 receptor from both rats and humans (Surprenant et al., 1996
Channel permeability and pore formation
Usually, P2X receptors are described as nonspecific cation channels permeable for Na+ and K+ as well as for Ca2+ (Soto et al., 1997
Moreover, the pore formation may not only differ among species but may also depend on the cell type. Humphreys et al. (1998)
Functional implications
Müller cells may be exposed to extracellular ATP in different ways. It has long been known that ATP is released into the mammalian eye by stimulation of the trigeminal nerve (Maul and Sears, 1979
In any case, it can be stated that human Müller cells express functional P2X7 receptors, which mediate rather small cation currents that are inwardly directed at resting membrane potential and can produce depolarizations. Activation of these receptors causes, at best, moderate Ca2+ influxes but no opening of large-conductance pores. Preliminary experiments on Müller cells from pathologically altered retinae indicate that there is no significant increase in the probability of pore formation when compared with cells from healthy retinae (T. Pannicke and F. Faude, unpublished data). It is therefore highly improbable that the P2X7 receptors of human Müller cells may act as "suicide triggers" as proposed for other cell types (Ferrari et al., 1997
One of the few ATP effects studied on Müller cells is a net "release" of GABA by reducing GABA uptake after activation of P2X receptors (Neal et al., 1998
FOOTNOTES Received March 24, 2000; revised May 23, 2000; accepted June 1, 2000.
This work was supported by the Bundesministerium für Bildung, Forschung und Technologie (BMB+F), Interdisciplinary Center for Clinical Research at the University of Leipzig (01KS9504, Project C5), by Deutscher Akademischer Austauschdienst (DAAD) 313/ARC-pz, and by the Deutsche Forschungsgemeinschaft (PA 615/1-1, AL 414/31). We thank Dr. Knut Krohn (Interdisciplinary Center for Clinical Research at the University of Leipzig, Grant Z3) for sequencing the DNA fragment, Dr. Dagmar Müller for helpful discussion of the single-cell PCR experiments, Dr. Ute Gröschel-Stewart for methodological support, and Roy Jordan for critical reading of this manuscript.
Correspondence should be addressed to Dr. Thomas Pannicke, Paul-Flechsig-Institute for Brain Research, Department of Neurophysiology, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany. E-mail: pant{at}medizin.uni-leipzig.de.
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