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channels in pigmented ciliary
epithelial cells
Departments of 1 Physiology and 3 Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Purines regulate intraocular pressure. Adenosine activates
Cl
channels of nonpigmented ciliary epithelial cells
facing the aqueous humor, enhancing secretion. Tamoxifen and ATP
synergistically activate Cl channels of pigmented ciliary
epithelial (PE) cells facing the stroma, potentially reducing net
secretion. The actions of nucleotides alone on Cl channel
activity of bovine PE cells were studied by electronic cell sorting,
patch clamping, and luciferin/luciferase ATP assay. Cl
channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 µM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2,
and cAMP activated Cl channels without enhancing effects
of 100 µM ATP. Buffering intracellular Ca2+
activity with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid
or blocking PGE2 formation with indomethacin
inhibited ATP-triggered channel activation. The Rp stereoisomer
of 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited protein
kinase A activity but mimicked 8-bromoadenosine 3',5'-cyclic
monophosphate. We conclude that nucleotides can act at >1 P2Y
receptor to trigger a sequential cascade involving Ca2+,
PGE2, and cAMP. cAMP acts directly on Cl
channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.
aqueous humor formation; P2Y receptors; adenosine 3',5'-cyclic monophosphate; prostaglandins; calcium
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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AQUEOUS HUMOR SECRETION is a determinant of intraocular pressure, so that reducing the secretory rate is a major strategy in treating glaucomatous patients. The aqueous humor also delivers substrates, oxygen, and the antioxidant ascorbate to the avascular cornea, lens, and trabecular meshwork, removes metabolic waste products, and facilitates immune responses (20). The bilayered ciliary epithelium forms aqueous humor by transferring solute (and, secondarily, water) from the stroma of the ciliary processes to the contralateral posterior chamber of the eye. Solute is taken up from the stroma by the pigmented ciliary epithelial (PE) layer, passes through gap junctions to the nonpigmented ciliary epithelial (NPE) layer, and is then released into the aqueous humor.
Several lines of evidence suggest that Cl channel
activity limits the rate of secretion (7). Activating
Cl channels of the NPE cells is expected to increase
secretion, whereas activating Cl channels of the PE cells
should favor reabsorption, thereby reducing net secretion. Purines may
regulate activity of Cl channels on both sides of the
tissue. At the aqueous surface of the epithelium,
A3-subtype adenosine agonists activate Cl
channels (5, 25). At the stromal surface, ATP and the
estrogen receptor antagonist tamoxifen synergistically activate
Cl channels of bovine PE cells (26). The aim
of the present study was to examine the effect of ATP itself on these cells.
The strategy of the study was to focus on ATP-triggered transfer of
fluid out of PE cells with volumetric measurements and to verify the
role of Cl channels by patch clamping. These approaches
were supplemented by luciferin/luciferase assay of ATP release in
addressing the identity of the nucleotide receptor(s) involved.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cellular model: Bovine PE cells. We have extended our studies of an immortalized PE cell line developed by M. Coca-Prados from a primary culture of bovine PE and characterized by several investigators (10, 26, 34). Cells were grown in Dulbecco's modified Eagle's medium (DMEM; no. 11965-084, GIBCO-BRL, Grand Island, NY) with 10% fetal bovine serum (SH30071.03, HyClone Laboratories, Logan, UT) and 50 µg/ml gentamicin (no. 15750-060, GIBCO-BRL), at 37°C in 5% CO2 (36). The medium had an osmolality of 328 mosmol/kgH2O. Cells were passaged every 6-7 days.
Volumetric measurements and analysis. After reaching confluence, cells from a T-75 flask were harvested by trypsinization within 3-10 days after passage (8). A 0.5-ml aliquot of the cell suspension in DMEM was added to 20 ml of each test solution. Parallel aliquots of cells were studied on the same day. One or two aliquots served as control, and the others were exposed to different experimental conditions at the time of suspension. The same amount of solvent vehicle was always added to the control and experimental aliquots. The sequence of studying the suspensions was varied to preclude systematic time-dependent artifacts. Cell volumes of isosmotic suspensions were measured with a Coulter counter (model ZBI-Channelyzer II) with a 100-µm aperture. As previously described, the cell volume (vc) of the suspension was taken as the peak of the distribution function. The time course of cell shrinkage was fit to a monoexponential by nonlinear least-squares analysis, and the probability of the null hypothesis (that any 2 sets of observations were derived from the same population) was obtained from the F distribution (10).
Whole cell patch-clamp recording.
Micropipettes were pulled from Corning no. 7052 glass, coated with
Sylgard, and fire polished. The resistances of the micropipettes in the
bath usually ranged from ~1.0 to 2.6 M
; successful seals displayed
gigaohm resistances. Unless otherwise stated, currents were recorded in
the ruptured-patch mode. After rupture of the membrane patch, the
series resistance was measured to be only 8.1 ± 0.7 M and was
therefore not usually compensated; whole cell capacitance was 10.4 ± 1.2 pS. The baseline whole cell currents were 83 ± 17 pA/pF.
In a subset of experiments (n = 8), we measured whole
cell currents in the perforated-patch mode. In those experiments, we
back-filled the micropipettes with solution containing amphotericin (168 µg/ml) and filled the tips with amphotericin-free solution (1). The applied voltages were not corrected for the small junction potentials (approximately 
2.8 mV; Ref. 6)
arising from the present micropipette filling and external solutions.
40 mV and stepped to test voltages from 100 to +80 mV
in 20-mV increments at 1-s intervals. Each step lasted 300 ms with
intervening periods of 1.7 s at the holding potential. Stimulatory
responses were measured at peak levels and inhibitory responses at the nadirs.
ATP measurements. Bovine PE cells were grown for 4-48 h to confluence on glass coverslips. Cells were washed in control solution and mounted on an inverted microscope, and bath ATP levels were measured continuously by including 2 mg/ml of luciferin/luciferase assay mixture (33). After background levels were recorded, a solution containing luciferin/luciferase assay mixture and either control solution or UTP was carefully added to the cells. ATP released from cells into the extracellular bath reacted with the luciferase and led to the production of a photon. Light produced was captured with a ×20 objective, filtered at 520 nm, measured with a photomultiplier tube, and recorded on-line using the Felix software suite (Photon Technologies International, Princeton, NJ). The luminescence values were converted to concentrations of ATP using a standard curve; UTP did not alter luminescence in the presence of the luciferin/luciferase assay mixture alone. The control solution contained (in mM) 105 NaCl, 4.5 KCl, 2.8 Na-HEPES, 7.2 HEPES acid, 1.3 CaCl2, 0.5 MgCl2, 5 glucose, and 75 mannitol. The pH was adjusted to 7.4 with NaOH, and the solution had an osmolality of 304-312 mosmol/kgH2O.
Chemicals. All chemicals were reagent grade. Gramicidin D, ionomycin, tamoxifen, ATP, UTP, ADP, UDP, GTP, 2-methylthio-ATP, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), dibutyryl cAMP (DBcAMP), and indomethacin were purchased from Sigma (St. Louis, MO); 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) from Molecular Probes (Eugene, OR); and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) from Biomol Research Laboratories, (Plymouth Meeting, PA). 9-Phenylanthranilic acid (DPC) was obtained from Fluka (Ronkonkoma, NY), and the inhibitory (Rp) and stimulatory (Sp) diastereoisomers of 8-bromoadenosine 3',5'-cyclic monophosphothioate (8-Br-cAMPS) were from Biolog (La Jolla, CA).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of nucleotides on PE cell volume.
ATP triggered concentration-dependent shrinkage of PE cells at 10 and
100 µM but not at 3 µM (Fig.
1A). Test solutions in Fig.
1A contained the cation ionophore gramicidin (5 µM) to
incorporate an exit port for K+ in the plasma membrane.
Under these conditions, the observed shrinkage reflected activation of
a Cl release pathway (8). ATP also produced
comparable shrinkage without gramicidin (Fig. 1B). As
previously reported (26), ATP did not uniformly trigger
shrinkage; no response was noted in ~20% of the present series of
volumetric measurements. Averaging the results of 11 series of
experiments (reflecting 43 experiments), ATP produced a magnitude of
shrinkage (
v
) of 4.2 ± 0.3% with a
time constant (
) of 5.2 ± 0.6 min. In controls (14 series, 50 experiments), v = 1.3 ± 0.3%
and = 9.5 ± 2.5 min (in 10 series of controls with
significant shrinkage). In agreement with our previous study
(26), tamoxifen uniformly enhanced the response to ATP
(n = 10, Fig.
2B). In contrast, adenosine
has no significant effect on the volume of these PE cells with or without tamoxifen (26).
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channel
blockers. When tested in parallel aliquots of cell suspensions, NPPB (100 µM) and DPC (1 mM) were similarly effective in blocking
shrinkage (Fig. 1B).
UTP and UDP (Fig. 2A) and ADP (Fig. 2B) also
triggered shrinkages. These effects were smaller than the ATP-triggered
shrinkage, precluding a definitive ranking of the effects of ADP, UDP,
and UTP.
Effect of nucleotides on PE whole cell currents.
ATP altered whole cell currents in approximately one-third of the
bovine PE cells (see Table 2). Two different electrophysiological effects were noted, sometimes in the same cells (Fig.
3). The stimulatory effect is exemplified
by the increase in whole cell currents beginning ~1 min after
initiation of perfusion with 10 µM ATP (Fig. 3A). Raising
the external ATP concentration to 100 µM then triggered the second
characteristic effect, a small inhibition of outward current at +80 and
+60 mV within ~40s. The later rate of increase in whole cell currents
was not detectably altered by this increase in ATP concentration.
Application of the Cl channel blocker NPPB (100 µM)
subsequently inhibited the currents by ~70%. The time courses of
ATP-difference currents after step changes in voltage are presented in
Fig. 3B, and the current-voltage relationships of the ATP-
and NPPB-difference currents are presented in Fig. 3C.
Slight inactivation was noted at highly depolarizing potentials (Fig.
3B); the magnitude of depolarization-induced inactivation
displayed by Cl channels of many cells depends on the
free intracellular Mg2+ concentration and other
unidentified factors (29). The current-voltage relationship was outwardly rectifying (Fig. 3C).
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26.8 ± 1.8 mV. Taking into account a
junction potential of approximately 2.8 mV estimated for similar
filling and bath solutions (5), the corrected reversal
potential was 29.6 mV. When this value and the known anionic
concentrations inside and outside the cell (Table
1) are inserted in the Goldman equation, the relative permeability of aspartate/Cl of the
ATP-activated anion channels can be estimated (5) to be
~0.14.
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33.4 ± 1.2 mV; Fig. 4C). As for the ATP experiments, the magnitudes of
the responses to UTP are displayed in Fig.
5 and the frequency of the responses is
given in Table 2. ADP and UDP triggered
similar changes, but the magnitudes were smaller (Fig. 5, Table 2).
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channels.
Because <50% of the cells responded to nucleotides, we wondered
whether dialysis of important components out of the cell could have
limited the frequency of response. However, the response rate to ATP
and UTP was not significantly enhanced by using the perforated-patch mode of whole cell recording. Stimulatory and inhibitory responses were observed in 5 and 3 of 11 cells, respectively.
Second messenger cascade assayed by shrinkage.
ATP increases intracellular Ca2+ activity of bovine PE
cells (26). Raising intracellular Ca2+ levels
by adding ionomycin triggered PE cell shrinkage similar to that
produced by ATP, and the two effects were not additive (Fig.
6A).
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channels, and thereby shrinkage, by stimulating increases in
Ca2+, PGE2, and cAMP, but it was unclear
whether these second messengers were acting in parallel or in tandem,
as recently found in Madin-Darby canine kidney (MDCK) cells
(30). We addressed this issue by attempting to block
increases 1) in Ca2+ (with the Ca2+
buffer BAPTA), 2) in PGE2 (with the
cyclooxygenase inhibitor indomethacin), and 3) in
cAMP-activated protein kinase activity (with the inhibitory Rp
stereoisomer of 8-BrcAMPS).
Preincubation with BAPTA-AM for 1 h to buffer intracellular
Ca2+ (18) exerted no direct effect on cell
volume but completely abolished ATP-triggered shrinkage (Fig.
7A). In contrast, similar treatment with TPEN, a
chelator of heavy metals other than Ca2+ and
Mg2+, did not alter the subsequent response to ATP (Fig.
7A). Even in the presence of BAPTA-AM, both 10 µM
PGE2 and 500 µM 8-BrcAMP triggered shrinkage (data not
shown; n = 4; P < 0.01).
We then examined whether blocking the cyclooxygenase pathway of
arachidonic acid metabolism would also affect the response to ATP. As
illustrated by Fig. 7B, both 1 and 100 µM indomethacin inhibited the ATP-triggered shrinkage. The indomethacin was not acting
simply as a channel blocker. Suspending cells in solution containing 10 µM PGE2 together with 100 µM indomethacin and 100 µM
ATP overcame the indomethacin inhibition (data not shown;
n = 5; P < 0.01 compared with parallel
aliquots suspended in indomethacin and ATP alone).
In our third approach, we focused on cAMP in trying to interrupt the
signaling pathways initiated by ATP. In a preliminary test, we found
that the specific form of cAMP used to activate shrinkage was not
critical. Effects similar to those elicited by 8-BrcAMP, albeit faster,
were triggered by the more permeable analog DBcAMP (500 µM) (Fig.
8A). Also, simultaneous
addition of the two analogs produced the same response as did
DBcAMP alone (Fig. 8A), so that 500 µM is maximally
effective. Because cAMP commonly modifies channel activity through
protein kinase A (PKA), we applied the inhibitory diastereoisomer
(Rp) of 8-BrcAMPS. Unexpectedly, Rp-8-BrcAMPS produced cell
shrinkage comparable to that triggered by ATP, and adding Rp-8-BrcAMPS
and ATP together had the same effect as adding ATP alone (Fig.
8B). Parallel additions of the inhibitory diastereoisomer,
the stimulatory isomer Sp-8-BrcAMPS, and 8-BrcAMPS in the same series
of experiments elicited similar shrinkages (Fig. 8C).
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Second messenger cascade assayed by whole cell currents.
Perfusion with 10 µM PGE2 activated Cl
currents in five of five cells by 88 ± 27% (+80 mV;
P < 0.01). In the experiment shown in Fig.
9A, perfusion with either 1 mM
DPC or 100 µM NPPB reversibly inhibited the activated currents and
isotonic washout was associated with decay of the currents. The time
courses of the PGE2-difference currents after step changes
in voltage are displayed in Fig. 9B, and the current-voltage
relationships are presented in Fig. 9C. With 100 µM
indomethacin present to block PGE2 production, 100 µM ATP
produced no stimulatory response (n = 3), and,
conversely in the presence of 100 µM ATP, indomethacin reduced
current by 31 ± 8% (+80 mV; n = 4;
P < 0.05); one-half of these cells displayed a
stimulatory response to the ATP pretreatment.
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channels were also examined
with whole cell patch clamping. In the experiment shown in Fig. 10A, 100 µM 8-BrcAMP
increased outward and inward currents. Raising the concentration to 500 µM further stimulated the currents, producing a cumulative
stimulation of ~80% at +80 mV. NPPB subsequently inhibited the
activated currents reversibly by >90%. The time courses of the
difference currents after step changes in voltage and the
current-voltage relationships are displayed in Fig. 10, B
and C, respectively. At concentrations of 100 and 500 µM,
8-BrcAMP stimulated currents in four of nine experiments without
exerting any blocking effect (Fig. 5).
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31.2 ± 3.3 mV in Fig. 9C and 32.7 ± 2.0 mV in Fig. 10C).
P2 receptors.
If a single P2 receptor modulated Cl channel activity in
PE cells, the data of Figs. 2 and 5 might point to P2Y11
(see DISCUSSION). Because the bovine sequence for this
receptor is unknown, we conducted a functional test for its presence.
The EC50 values of 2-methylthio-ATP at turkey
P2Y1 and human P2Y11 receptors are 6 nM and 50 µM, respectively (17). However, neither 100 nM nor 100 µM concentrations of 2-methylthio-ATP triggered the shrinkage
produced by 100 µM ATP (n = 4; data not shown).
ATP release by PE cells.
Multiple complexities limit the identification of P2 receptors
affecting cell functions (see DISCUSSION). One such
complexity is potential UTP-triggered ATP release, which has been
reported in other cells (9, 23, 30). This possibility was
examined by perfusing PE cells with 100 µM UTP. As illustrated by
Fig. 11, the mechanical perturbation
associated with solution change triggered a transient release in ATP
even in the control solution. However, UTP triggered a sustained
release of ATP. At the peak, 7 min after solution change, ATP levels in
control solution were 41.1 ± 9.4 nM whereas those in UTP were
206.8 ± 73.5 nM (P < 0.05; n = 10). The subsequent decline in detected external ATP concentration likely reflects the activity of ecto-ATPase/apyrase and the utilization of substrate (23, 24).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Initial observation.
External nucleotides activated Cl channels and triggered
release of intracellular solute and water from bovine PE cells. On average, ATP produced larger stimulations of Cl currents
than did UTP, ADP, and UDP at the same concentration (Fig. 5). However,
the observation that more than half the cells did not respond and the
ranges in magnitude of the responses made it difficult to rank the
nucleotide responses from the patch-clamp data. In this respect,
electronic cell sorting provided a more feasible approach, directly
measuring the parameter of central interest (the transfer of fluid out
of the cells) and sampling 50,000 cells or more for each data point. By
this measure and at a concentration of 100 µM, the ranking is
ATP > UTP, ADP, UDP (Fig. 2).
conductance in 49% of the freshly dissected bovine PE cells they studied. Mitchell et al. (27) also found that GTP
S
activated two-thirds of large-conductance Cl channels in
patches taken from freshly dissected bovine PE cells ultimately shown
to possess such a channel. Given the syncytial nature of the bilayered
ciliary epithelium (7), activation of the Cl
channels in 25-45% of the PE cells should provide a
physiologically significant pathway for release of solute to the stroma.
Nucleotide receptors. Identification of functionally important receptor(s) is generally even more complex for P2 than for P1 (adenosine) receptors because (11, 21) 1) specific agonists and antagonists are not available; 2) the ectoenzymes apyrase, ecto-ATPase, ecto-ADPase, 5'-nucleotidase, and ectonucleoside diphosphokinase not only metabolize adenine nucleotides to adenosine but also interconvert purine and pyrimidine nucleotides; 3) cells frequently possess multiple P2 and P1 receptors, which can exert opposing effects; and 4) the final functional effects can be highly dependent on the specific cell studied because of synergistic interactions of the second messenger cascades. An additional caveat is illustrated by Fig. 11. Consistent with observations in other cells (9, 23, 30), application of one triphosphate nucleotide can trigger release of another nucleotide from intracellular stores. The concentrations of ATP measured directly in this study, although probably an underestimate, should be sufficient to stimulate the P2Y2 receptor with an EC50 of 200 nM (22).
Despite these caveats, two general conclusions can be drawn from the data. First, the dominant functional receptors must be P2Y metabolotropic, rather than P2X ionotropic receptors. Otherwise, ATP activation of P2X cation-nonselective channels would have triggered influx of cation, leading to swelling of the PE cells, contrary to observation. Second, it is unlikely that the nucleotide-triggered shrinkage was mediated by occupancy of a single population of P2Y receptors. Of the six cloned functional P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12; Refs. 11 and 14), only P2Y11 might conform to the nucleotide ranking displayed by Fig. 2. Our inability to detect a response to the P2Y11 agonist 2-methylthio-ATP at 100 µM suggests that this receptor is not playing a dominant role in mediating the responses to ATP. We conclude that the transport effects of the nucleotides likely reflect occupancy of multiple P2 receptors of the PE cells. This conclusion is consistent with the reported detection of at least two different P2Y receptors in bovine PE cells (31, 34).Second messengers.
The present data suggest a plausible signaling cascade. We have found
that elevation of intracellular Ca2+ activity and separate
application of PGE2 and 8-BrcAMP all mimic ATP in reducing
cell volume, and the effects of these agents are not additive with
those of ATP. In principle, all three second messengers could act in
parallel to mediate the ATP-triggered activation of Cl
channels (Fig. 12A).
Alternatively, ATP could trigger a cascade involving the sequential
activation of the three second messengers (Fig. 12B). More
complex pathways involving both parallel and series activations are
also possible.
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channels
(Fig. 10), permitting release of K+ and Cl
through parallel ionic channels and, secondarily, release of water
(Figs. 6C, 7C, 8A, and
8C). The data of Fig. 8, B and
C, obtained with the PKA-inhibitory analog of 8-BrcAMP,
Rp-8-BrcAMPS, suggest that cAMP acts directly on the PE
Cl channel, not through activation of PKA. This concept
is consistent with reports that Rp-cAMPS mimics cAMP-triggered
activation of hyperpolarization-activated currents (3, 15)
and Rp-cGMPS activates the photoreceptor (but not the olfactory) cyclic
nucleotide-gated channel (19).
Potential physiological implications.
The Cl channels of PE and NPE cells differ in their
unitary conductances (27, 37) and pharmacological profiles
(28, 35). In principle, activation of the NPE
Cl channels facing the aqueous humor is expected to
increase net secretion, whereas activation of PE channels facing the
stroma is expected to reduce net secretion. Release of ATP from both NPE and PE cells (24) provides the basis for autocrine
regulation of secretion. Ectoenzyme metabolism of ATP provides a source
of adenosine, which activates NPE Cl channels at ~3
µM concentration (5, 25). The results of the present
work demonstrate that ATP itself activates PE Cl channels
at ~10 µM concentration (Figs. 1 and 3). Which effect predominates
should depend on local purine concentrations, on ectoenzyme activities
and receptor densities at both surfaces, and possibly on activities of
additional modulators. In particular, occupancy of plasma membrane
estrogen receptors is thought to trigger synergistic enhancement of ATP
activation of PE cell Cl channels (35). The
basis for the synergism between the estrogen-triggered cascade and the
Ca2+-PGE2-cAMP cascade triggered by ATP remains
to be determined.
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ACKNOWLEDGEMENTS |
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We thank Dr. Martin Pring for suggestions concerning statistical analysis of the data and Dr. Jeffrey W. Karpen for helpful information.
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FOOTNOTES |
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This work was supported in part by National Eye Institute Grants EY-12213, EY-08343, and EY-01583 (for core facilities). J. C. Fleischhauer received support from Swiss National Science Foundation Fellowship No. 1037.
Address for reprint requests and other correspondence: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 February 2001; accepted in final form 26 June 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) and activated (
) currents and 100 µM ATP (
)- and 100 µM NPPB
(
)-difference currents.
