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1 From the Department of Neurophysiology, Paul Flechsig Institute of Brain Research, and the 2 Department of Ophthalmology, Eye Hospital, University of Leipzig, Germany.
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Abstract |
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METHODS. Human Müller cells were enzymatically isolated from donor retinas, and whole-cell patch-clamp recordings were made to characterize the density of the P2X7 currents and the activation of currents through Ca2+-activated K+ channels of big conductance (IBK) that reflects the increase of the intracellular Ca2+ concentration.
RESULTS. Stimulation by external ATP or by benzoylbenzoyl ATP (BzATP) evoked both release of Ca2+ from thapsigargin-sensitive intracellular stores and opening of Ca2+-permeable P2X7 channels. These responses caused transient and sustained increases in IBK. In Müller cells from patients with PVR, the mean density of the BzATP-evoked cation currents was significantly greater compared with cells from healthy donors. As a consequence, such cells displayed an enlarged IBK during application of purinergic agonists. ATP and BzATP increased the DNA synthesis rate of cultured cells. This effect could be reversed by blocking the IBK.
CONCLUSIONS. The increased density of P2X7 receptor channels may permit a higher level of entry of extracellular Ca2+ into cells from patients with PVR. Enhanced Ca2+ entry and the subsequent stronger activation of IBK may contribute to the induction or maintenance of proliferative activity in gliotic Müller cells during PVR.
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Introduction |
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Müller glial cells express a diversity of receptors for
neurotransmitters and other biologically active
substances6
that may modulate the membrane conductances of
the cells.7
Adenosine triphosphate (ATP) is an important
transmitter in the retina,8
being crucially involved in
early retinal development9
10
and in the neuronal
information processing of the mature retina.11
Müller cells may express different types of purinergic P2
receptors. In isolated salamander Müller cells and in rat
Müller cells in situ, activation of P2Y receptors by
extracellular ATP stimulates the release of Ca2+
from internal stores.12
13
Activation of P2 receptors
inhibits the uptake of 
-aminobutyric acid (GABA) by rat Müller
cells.14
Recently, the presence of ionotropic P2X
receptors was described in Müller cells freshly isolated from the
human retina.15
In human Müller cells, extracellular
ATP and 2'-/3'-O-(4-benzoylbenzoyl)-ATP (BzATP), a more
specific agonist of P2X7 receptors,16
open
nonselective cation channels that may be permeable for
Ca2+ ions.15
The noninactivating
current kinetics, as well as single-cell reverse
transcription–polymerase chain reaction (RT-PCR) and
immunocytochemical evidence, indicate the expression of
P2X7 receptors in these cells.15
Because the activation of purinergic receptors is thought to be a mediator of the induction of reactive gliosis,17 18 19 we wanted to investigate whether the expression of P2 receptors by human Müller cells is altered under pathologic conditions. For this purpose, the density of BzATP-evoked currents in cells from patients with PVR and those with choroidal melanoma was compared with that in cells from healthy donors. Alterations of cell membrane conductances during activation of P2 receptors were investigated electrophysiologically in two ways: Either the P2X7 receptor-mediated cation conductance or the stimulation of the Ca2+-activated K+ currents were recorded. An amplitude increase of the Ca2+-activated K+ currents reflects the increase of the intracellular Ca2+ concentration induced by activation of the purinergic receptors.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Electrophysiological Recordings
Records were made in the whole-cell or in the excised patch
configuration of the patch-clamp technique.21
To
create outside–out patches, the whole-cell configuration was
established, and thereafter the pipette was drawn back to excise a
membrane patch. Voltage-clamp records were performed at room
temperature (22°C–25°C) using an amplifier (EPC 7; List,
Darmstadt, Germany) and a computer program (Tida ver. 5.72, Heka
Elektronik, Lambrecht, Germany). The signals were low-pass filtered at
4 kHz (three-pole Bessel filter) at a sampling rate of 15 kHz. The
series resistance (10–18 M
) was compensated by 30% to 50%. Patch
pipettes were pulled from thick-walled borosilicate glass (WPI,
Sarasota, FL) and had resistances between 3 and 5 M when
K+-containing bath and pipette solutions were
used. To investigate ATP-evoked responses, the whole-cell currents were
elicited by a standard step protocol (holding potential,
Vh -80 mV; depolarizing and
hyperpolarizing voltage steps of 250-msec duration with an increment of
20 mV) or by continuous recording at a
Vh of -60 mV with voltage steps of 50
msec duration to +120 mV and to -100 mV at a frequency of 2.5 Hz. The
traces were not leak subtracted. Data were not corrected for liquid
junction potentials, because these did not exceed 3 mV. The membrane
capacitance of the cells was measured by the integral of the
uncompensated capacitive artifact evoked by a hyperpolarizing voltage
step from -80 to -90 mV when Ba2+ ions (1 mM)
were present in the bath solution to block the K+
conductance. For recording of the capacitive artifact, the sampling
rate was 30 kHz, and the frequencies above 10 kHz were cut off.
Solutions
The recording chamber was continuously perfused with bath
solution. Test substances were added by fast (<15 seconds) changes of
the perfusate. For recording the effects of purinergic agonists on the
whole-cell currents and on the BK channel activity in excised membrane
patches, a low-divalent cation bath solution was used composed of (mM)
110 NaCl, 3 KCl, 0.5 CaCl2, 10 HEPES, and 11
glucose with pH adjusted to 7.4 with Tris. The pipette solution was
made of (mM) 10 NaCl, 130 KCl, 3 MgCl2, 0.1 EGTA,
and 10 HEPES with pH adjusted to 7.2 with Tris. When the BzATP-induced
cation currents were recorded in K+-free
conditions, the bath solution consisted of (mM) 116 NaCl, 1
Na2HPO4, 25
NaHCO3, 11 glucose, 10 HEPES (pH 7.4), and was
gassed with 95% O2-5%
CO2. The pipette solution contained (mM) 10 NaCl,
130 CsCl, 1 CaCl2, 2 MgCl2,
10 EGTA, 10 HEPES (pH 7.1). Iberiotoxin was obtained from Alomone
Laboratories (Jerusalem, Israel) and papain from Boehringer–Mannheim
(Mannheim, Germany). All other substances were from Sigma (Deisenhofen,
Germany).
Cell Culture
Primary cultures of Müller cells were obtained from
retinas of healthy donors. The excised retinas were dispersed in
Ca2+, Mg2+-free phosphate
buffer supplemented with nagarse (1 mg/ml) for 30 minutes at 37°C.
After they were washed in phosphate buffer containing DNase I (200
U/ml), the dissociated cells were seeded on coverslips (100 µl cell
suspension per coverslip; the retinal cells from two eyes were
distributed on 54 coverslips) and cultured at 37°C in a gas mixture
of 95% air-5% CO2. The minimum essential medium
was supplemented with 10% fetal calf serum. The medium was exchanged
twice a week. After 3 weeks in culture, the test substances were added
to the culture medium 16 hours before the cultures were fixed. During
this latter period, substances were tested in serum-free medium.
Lipophilic substances were dissolved in dimethyl sulfoxide (DMSO).
Vehicle alone did not affect the DNA synthesis rate.
Determination of the DNA Synthesis Rate
The DNA synthesis rate was determined by measuring the
bromodeoxyuridine (BrdU) incorporation. BrdU (10 µM) was added 16
hours before fixation with 4% paraformaldehyde. BrdU incorporation
into nuclei of mitotically active cells was revealed by a murine
anti-BrdU IgG-antibody (Bu 33; Sigma) and Cy3-tagged secondary
antibodies. Counter-labeling of all cell nuclei was performed with
acridine orange or Hoechst 33258. In the peripheral (i.e.,
nonconfluent) regions of the cultures, six distinct areas of each
coverslip (each approximately 60,000
µm2, resulting in a total area of 0.42
mm2 per coverslip) were studied by means of a
semiautomatic image analysis system (SIS; Soft-Imaging Systems,
Münster, Germany). The results from three coverslips per culture
were summarized. The experiments involved four independent cultures.
The ratio of BrdU immunoreactive versus total cell nuclei was taken as
marker for the DNA synthesis rate.
Data Analysis
The steady state whole-cell currents were measured at the end of
250-msec voltage steps. To determine disease-related changes of
currents, 6 to 13 cells per donor were recorded; in most of the further
statistical analysis, only the mean values of the cells from each donor
were used. Statistical analysis (Mann–Whitney test, two-tailed;
nonparametric regression analysis) and curve fits were made by computer
(Prism; GraphPad, San Diego, CA). Data are expressed as means ±
SD (electrophysiological data) or as means ± SEM (proliferation
experiment).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Similar results were obtained with external application of BzATP. As illustrated in Figure 1E , BzATP (50 µM) reversibly increased both inward and outward currents of Müller cells from patients with PVR, whereas the outward currents at positive potentials were much more increased than the inward currents at negative potentials. Moreover, BzATP depolarized the cells, as indicated by the positive shift of the zero current potential of the whole-cell currents (by 9.6 mV; inset in Fig. 1E ). The BzATP-induced outward currents displayed significantly different amplitudes when the drug was tested in Ca2+-containing or in Ca2+-free bath solution. In Ca2+-containing bath solution, the BzATP-induced current had a mean density of 17.3 ± 10.1 pA/pF (n = 7; measured at the voltage step to +140 mV), whereas in Ca2+-free solution the current density was only 3.3 ± 4.2 pA/pF (n = 8, P < 0.01). It is concluded that a large portion of the BzATP-induced outward current was evoked by Ca2+ entry from the extracellular space and may represent the activation of Ca2+-activated K+ currents. A similar increase of depolarization-evoked, Ca2+-activated K+ currents was observed in cells from healthy donors (Fig. 3) .
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To determine whether the outward current elevated by BzATP represents a BK-channel–mediated current (IBK), the effect of iberiotoxin was tested. Figure 3A illustrates an example of current records in one Müller cell from a healthy donor. Extracellular application of BzATP (50 µM) induced a strong increase of the outwardly directed currents that was blocked by simultaneous exposure to iberiotoxin (100 nM). The time course of the drug’s effect is shown in Figure 3B for one cell. Iberiotoxin fully reversed the BzATP-induced increase of the outward currents at +120 mV but had no effect on the inward currents at -100 mV. The voltage-dependence of the whole-cell currents reveals that BzATP induced a negative shift of the activation of IBK; under control conditions, the activation threshold of the IBK was at +120 mV, and during BzATP exposure, the IBK was evoked at potentials positive to 0 mV (Fig. 3C) . Iberiotoxin blocked the BzATP-activated IBK measured at +120 mV (Fig. 3D , left), but did not influence the amplitude of the BzATP-induced inwardly directed currents measured at -100 mV (Fig. 3D , right). These results indicate that activation of P2 receptors enhances the amplitude of IBK in human Müller cells and that the depressing effect of iberiotoxin on IBK was not caused by inhibition of the ATP-induced cation conductance.
In excised outside–out patches, BzATP reversibly increased the activity of iberiotoxin-sensitive (Fig. 4A ) K+ channels of large conductance (134.3 ± 16.6 pS). As shown in Figure 4B , exposure to BzATP (50 µM) of the extracellular side of the membrane increased the open probability of BK channels at positive membrane potentials. In three patches, BzATP increased the mean channel-open probability at +80 mV from 0.04 ± 0.05 to 0.14 ± 0.10. After washout of the drug, the value returned to 0.04 ± 0.04.
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Taken together, the results indicate that extracellular ATP may have three effects on human Müller cells: (1) Through activation of P2X7 receptors, ATP evokes the opening of a nonselective cation conductance which mediates a Ca2+ entry from the extracellular space and which depolarizes the cells; (2) Ca2+ ions are released from intracellular stores, probably through an activation of P2Y receptors; and (3) the depolarization and the elevation of the intracellular Ca2+ concentration together increase IBK.
Disease-Related Changes of BzATP-Evoked Currents
To determine whether P2 receptors of the Müller cells are
implicated in transdifferentiation processes accompanying gliosis in
cases of human retinal disease, two kinds of retinal diseases were
investigated: choroidal melanoma and PVR. Müller cell gliosis is
characterized by different features, including upregulation of the
immunoreactivity of intermediate filaments and by cell hypertrophy. In
the case of PVR, Müller cells may also become proliferative,
whereas in other types of gliosis (e.g., during choroidal melanoma),
Müller cells do not proliferate. Müller cells from patients
with PVR displayed a significantly greater cell membrane capacitance
(81.5 ± 22.4 pF, n = 19 patients) compared with cells
from healthy retinas (54.3 ± 10.9 pF, n = 13 donors,
P < 0.001) indicating hypertrophy of Müller
cells in PVR retinas. Similarly, Müller cells from three patients
with melanoma displayed hypertrophy (mean membrane capacitance,
93.0 ± 12.3 pF, n = 3).
Whole-cell currents were recorded before and during exposure to BzATP (50 µM) in K+-containing or K+-free solutions. Figure 5A shows the mean steady state current density–voltage relations of BzATP-evoked currents measured in K+-containing solutions after the transient stimulations of IBK had ceased (in most cells, 2 to 3 minutes after the beginning of drug exposure; currents were calculated by subtraction of the control currents from the drug-induced currents). Mean curves from three donor groups are shown: healthy donors, patients with PVR, and patients with melanoma. The BzATP-evoked currents consist of two components: At negative membrane potentials, the inwardly (downwardly) directed currents reflect the activation of the BzATP-evoked cation conductance, and, at positive potentials, the outwardly directed currents reflect the amplitude increase of the sustained IBK. The density of the inwardly directed cation currents evoked by BzATP was significantly greater in cells from patients with PVR (2.9 ± 1.1 pA/pF, measured between -100 and -140 mV) than in cells from healthy donors (1.1 ± 0.6 pA/pF, P < 0.001). The elevated amplitude of the inwardly directed currents was accompanied by a stronger stimulation of IBK at positive membrane potentials. However, in cells from the patients with melanoma, both the BzATP-evoked inward currents (0.53 ± 0.25 pA/pF) and the BzATP-stimulated IBK were in the same range as found in cells from healthy donors.
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Figure 5B shows mean values of the amplitudes of the maximally BzATP-stimulated IBK from the three donor groups. Although the amplitudes of the transient IBK were similar in all groups, the amplitude of the sustained IBK was significantly greater in cells from patients with PVR compared with healthy donors. This indicates a specific upregulation of P2X7 receptor-mediated Ca2+ entry, whereas the P2Y receptor-mediated release of intracellular Ca2+ was not altered in pathologic conditions.
Figures 5C and 5D show the voltages at which the sustained IBK was half-maximally activated, and the densities of the maximal IBK, respectively, for cells from diseased and healthy retinas. Both parameters were significantly correlated with the density of the BzATP-evoked cation conductance. The higher the density of the BzATP-evoked inward currents, the more shifted the voltage at which IBK was half-maximally activated toward more negative membrane potentials, and the higher the maximal density of stimulated IBK. Probably, this was mediated by the intracellular concentration of Ca2+. We did not find any correlation of the density of the ATP-gated cation currents with the age of the donors (not shown).
Modulation of the DNA Synthesis Rate by Extracellular ATP
Both extracellular Na-ATP (500 µM) and BzATP (20 µM) increased
the DNA synthesis rate of cultured human Müller cells (Fig. 6A
). Simultaneous exposure to iberiotoxin (70 nM) decreased the effects
of the purinergic agonists indicating an involvement of BK channel
activity in mediating the proliferation-stimulating effect of
extracellular ATP.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Purinergic Receptors of Müller Cells
In human Müller cells, both ATP and BzATP activate a
nonselective, noninactivating cation current,15
consistent
with the involvement of P2X7 receptors. The
nonselective cation channels allow for an entry of
Ca2+ ions from the extracellular
space15
that causes a sustained increase of
IBK (Fig. 1D)
. ATP and BzATP caused
also transient elevations of IBK that
persisted in Ca2+-free extracellular solution
(Fig. 2A)
, indicating that they induced a release of
Ca2+ from thapsigargin-sensitive intracellular
stores. Although BzATP is assumed to preferentially activate ionotropic
P2X7 receptors the present results indicate that BzATP also
stimulates internal Ca2+ release, as previously
described for other cell types.25
The mechanism of the
BzATP-induced release of internal Ca2+ remains to
be identified. This may be a secondary step after activation of
P2X7 receptors or a direct (additional)
activation of P2Y receptors by BzATP, as previously shown for
P2Y2 receptors.26
The presence of
P2Y receptors in human Müller cells was also indicated by
preliminary experiments that showed that, in addition to ATP,
extracellular uridine triphosphate (UTP) and guanosine triphosphate
(GTP) evoke a transient activation of
IBK (not shown).
Involvement of Purinergic Receptors in Müller Cell Gliosis
The involvement of purinergic receptors in induction or
maintenance of gliosis in vivo has been previously
discussed.17
19
In the brain, activation of P2 receptors
may induce astrogliosis, leading to hypertrophy and proliferation of
astrocytes.18
27
In the current study, we showed for the
first time that gliosis in vivo may be connected with an upregulation
of a distinct type of purinergic receptor. Although the currents
through P2X7 receptor channels were upregulated
in Müller cells from patients with PVR compared with healthy
donors, the release of intracellular Ca2+ that is
probably mediated by P2Y receptors was unchanged in these cells.
However, further investigations are necessary to provide evidence for a
causal relationship between the expression of
P2X7 receptor-mediated currents and the induction
or maintenance of Müller cell gliosis.
Although gliosis was present in eyes with choroidal melanoma, evidenced by a hypertrophy of the cells, all other investigated membrane conductances, including the density of P2X7 receptor currents, were in the range of the cells from healthy retinas. By contrast, gliosis accompanying PVR was characterized by hypertrophy, by a strong downregulation of IKir and a less negative resting membrane potential,5 20 by an increased expression of voltage-gated Na+ channels,28 and by a decrease of currents through voltage-gated Ca2+ channels,29 whereas the density of P2X7 receptor currents was upregulated. When data from all donor groups used in the present study were considered, an increase of the density of sustained BzATP-evoked inward currents was correlated with a decrease of the IKir density (r = -0.574, n = 31 donors, P < 0.001), a depolarization of the membrane (r = 0.596, P < 0.001), a decrease of the peak currents through high-voltage–activated Ca2+ channels (r = -0.600, P < 0.001), and a higher density of peak Na+ currents (r = 0.604, P < 0.001). As a mean, the more membrane features were altered the stronger the upregulation of P2X7 receptor-mediated currents by human Müller cells. This may implicate a causal relationship between purinoceptor activation and the strength of gliosis.
IBK and ATP-Induced Müller Cell
Proliferation
A specific upregulation of P2X7 receptor
currents in cells from patients with PVR may indicate that this type of
purinergic receptors is involved in processes that are activated when
gliotic Müller cells become proliferative. A role of
P2X7 receptors in induction and/or maintenance of
proliferation was previously described for
lymphocytes.30
31
Moreover, many tumor cell lines are
characterized by high P2X7 receptor expression
levels.32
We found that both ATP and BzATP stimulated the
DNA synthesis rate of cultured human Müller cells (Fig. 6A)
.
Coapplication of iberiotoxin fully reversed the effects of the
purinergic agonists. When we excluded an unspecific effect of
iberiotoxin on Müller cells, the data indicated that the
activation of IBK may be a step that
is necessary for the purinergic induction of Müller cell
proliferation. The mechanism of involvement of
IBK in the regulation of the
proliferation is unclear. One mechanism may be the regulation of the
strength of Ca2+ entry into Müller cells by
BK channels,33
as it was shown for the agonist-induced
Ca2+ entry in other cell types.34
Because elevated intracellular Ca2+ concentration
is necessary for both gliosis and maintenance of proliferative
activity, the increased expression of P2X7
receptors and the subsequent stronger activation of
IBK may promote the induction of both
processes in cells from PVR retinas. However, further experiments are
necessary to investigate the relationships between purinergic receptor
activation, BK current stimulation and induction and/or maintenance of
Müller cell proliferation.
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Footnotes |
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Submitted for publication September 7, 2000; revised November 15, 2000; accepted November 29, 2000.
Commercial relationships policy: N.
Corresponding author: Andreas Bringmann, University of Leipzig, Paul Flechsig Institute of Brain Research, Department of Neurophysiology, Jahnallee 59, D-04109 Leipzig, Germany. bria{at}server3.medizin.uni-leipzig.de
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). The records were made in bath solutions containing 0.5 mM
Ca2+ (left) or in
Ca2+-free bath solutions containing 1 mM EGTA
(right). Voltage steps were applied at a frequency of 2.5 Hz
from a holding potential of -60 mV (middle traces) to +120
mV (upper traces) and to -100 mV (lower traces;
inset). Dashed lines: Zero current levels. The
amplitudes were measured at the end of each of the 50-msec voltage
steps. Arrows: Beginning of drug exposure; horizontal
bars: 10 seconds; vertical bars: 250 pA. (B)
Mean amplitudes of the transient and sustained outward currents
activated by BzATP. The cells were examined in
Ca2+-free (n = 6) or in
Ca2+-containing bath solution (n = 7).
The amplitudes of the transient currents and of the sustained currents
were measured at the end of 50-msec voltage steps to +120 mV from a
holding potential of -60 mV and are calculated as the percentage of
the control currents that were measured 20 seconds before drug
application (100%). Significant differences of P <
0.05 (•) and of P < 0.01 (••). n.s., not
significant.
