Similarity of A3-adenosine and swelling-activated
Cl
channels in nonpigmented ciliary epithelial
cells
David A.
Carré1,
Claire H.
Mitchell1,
Kim
Peterson-Yantorno1,
Miguel
Coca-Prados2, and
Mortimer M.
Civan1,3
Departments of 1 Physiology and 3 Medicine, School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
and 2 Department of Ophthalmology and Visual Science, Yale
University School of Medicine, New Haven, Connecticut
06510
 |
ABSTRACT |
Chloride release from nonpigmented ciliary epithelial (NPE)
cells is a final step in forming aqueous humor, and adenosine stimulates Cl
transport by these cells. Whole cell patch
clamping of cultured human NPE cells indicated that the
A3-selective agonist
1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-
-D-ribofuranuronamide (IB-MECA) stimulated currents (IIB-MECA) by
~90% at +80 mV. Partial replacement of external Cl
with aspartate reduced outward currents and shifted the reversal potential (Vrev) from
23 ± 2 mV to
0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusion
with the Cl
channel blockers
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid
inhibited the currents. Partial Cl
replacement with
aspartate and NO3
, and perfusion with NPPB, had
similar effects on the swelling-activated whole cell currents
(ISwell). Partial cyclamate substitution for external Cl
inhibited inward and outward currents of both
IIB-MECA and ISwell. Both
sets of currents also showed outward rectification and inactivation at
large depolarizing potentials. The results are consistent with the
concept that A3-subtype adenosine agonists and swelling
activate a common population of Cl
channels.
aqueous humor secretion; anion selectivity; cyclamate; 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-
-D-ribofuranuronamide; MRS-1523
 |
INTRODUCTION |
THE AQUEOUS HUMOR OF THE
EYE is secreted by the bilayered ciliary epithelium
(9). The consensus view (4, 5,
13, 19, 20, 31,
34, 35) is that ions are taken up from the stroma of the ciliary processes by the pigmented ciliary epithelial cells, passed through gap junctions (8, 13,
15, 23, 25, 28) to
the nonpigmented ciliary epithelial (NPE) cell layer, and then released
into the aqueous humor. The water is thought to passively follow the
movement of these ions, mainly Na+ and Cl
,
across the epithelial tissue. One major factor likely to limit the rate
of secretion is the rate of release of Cl
into the
aqueous humor (6). Although the regulatory pathways of
Cl
channel activity are unclear, adenosine has been found
to activate NPE Cl
channels (3). The
potential importance of purinergic regulation is supported by the
association observed between activation of A2-adenosine and
A1 receptors with ocular hypertension (11) and
hypotension (10), respectively.
The physiological source of the external adenosine is likely to be the
ciliary epithelial cells themselves. NPE cells display reservoirs of
ATP both in culture and in the intact tissue, release ATP to the
extracellular fluid, and can metabolize the ATP to adenosine by their
ectoenzymes (21). Adenosine and its metabolite inosine can
rise to micromolar levels in the aqueous humor (12), a
concentration adequate for activating Cl
channels
(3).
The signaling pathway involved in adenosine activation of
Cl
channels is unknown. Recently, volumetric measurements
of cultured human NPE (HCE) cells in isotonic suspensions, coupled with
measurements of short-circuit current across rabbit ciliary epithelium
and RT-PCR amplification of RNA from cells and tissue, have suggested that adenosine activates Cl
efflux in NPE cells by
stimulating A3-subtype adenosine receptors (22). We have now studied the same human nonpigmented
ciliary epithelial cell line with whole cell patch clamping to examine whether Cl
channels are indeed activated by the
A3 agonist
1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-
-D-ribofuranuronamide (IB-MECA). We have also compared the anionic selectivity of
IB-MECA-activated and swelling-activated Cl
channels. The
results are consistent with the concept that A3 agonists
and swelling activate a common population of Cl
channels.
 |
MATERIALS AND METHODS |
Cellular model.
The HCE-immortalized nonpigmented ciliary epithelial cell line was
developed by M. Coca-Prados from primary cultures of human epithelium.
Cells were grown in DMEM (no. 11965-027; GIBCO BRL, Grand Island,
NY) containing 10% fetal bovine serum (A-1115-L; HyClone Laboratories,
Logan, UT) and 50 µg/ml gentamycin (no. 15750-011, GIBCO BRL) at
37°C in 5% PCO2 (37). The
medium had an osmolality of 328 mosmol/kgH2O. Cells were
passaged every 6-7 days and were studied within 6-10 days of
passage once confluence was reached.
In preparation of patch clamping, culture flasks were briefly
trypsinized (37) for 4.0-5.5 min. Cells were
resuspended in DMEM, plated onto the base of a plastic petri dish, and
allowed to settle and adhere for 15-20 min. A perforated lucite
insert was placed inside the petri dish to form the chamber contours and restrict the fluid volume to ensure rapid bath exchange.
Solutions.
The compositions of the internal filling solution of the
micropipette and the external bath solutions are presented in
Tables 1 and 2. The
internal solution contained 0.43 mM CaCl2 and 5.0 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to fix the free Ca2+ concentration at 10 nM. External and
internal solutions were adjusted to pH 7.4 and 7.2, respectively, by
addition of either NaOH or N-methyl-D-glucamine
(NMDG). The osmolalities of the isotonic bath and internal
solution were set at 295 ± 3 mosmol/kgH2O and 272 ± 3 mosmol/kgH2O, respectively. The
hypotonic bath solutions were made 60 mosmol/kgH2O
lower than the isotonic external solutions by suitable exclusion of
mannitol.
Ruptured-patch whole cell recording.
Micropipettes were double-pulled from Corning glass (no. 7052), coated
with Sylgard, and fire polished. The resistances of the micropipettes
in the bath ranged from 1.0 to 2.2 M
; successful seals commonly
displayed resistances of 20-50 G
. After rupture of the membrane
patch, the series resistance was measured to be 7.3 ± 0.8 M
.
All series resistances in excess of 5 M
were 80% compensated. In
the baseline NaCl-Ringer solutions of Tables 1 and 2, the baseline
whole cell currents were 51 ± 14 pA/pF and 46 ± 13 pA/pF, respectively.
All patches were formed in the high-Cl
-containing bathing
solutions (NaCl-Ringer solutions) of Tables 1 and 2. The ambient potentials in the bath were nulled to 0 mV before approaching the cell.
The junction potentials for the micropipettes relative to a flowing 3 M
KCl junction were measured to be 4.12 ± 0.02 mV and 2.76 ± 0.02 mV for the NaCl-Ringer solutions of Tables 1 and 2, respectively.
Additionally, account has been taken of the small change in junction
potential arising at the 3 M KCl reference junction when the bath
solution was changed after formation of the patches. Single junction
potentials cannot be measured but can be estimated with the Henderson
equation in the outside bath and in impaled cells (24).
These calculations were conducted with the Clampex 8.0 (Axon
Instruments) software program based on the approach of Barry and Lynch
(2). The relative mobility of cyclamate to K+
was estimated to be 0.24 from a comparison of the measured junction potentials in the presence of NaAsp- and NaCycl-Ringer solutions, using
the Clampex 8.0 program. The final corrections for junction potentials
ranged from 1.5 to 4.8 mV. These corrections have been applied in the
analysis of the measurements (as indicated in RESULTS and
DISCUSSION) but are not included in the figures, which
present the raw data.
Data were acquired at 1.25 kHz and filtered at 500 Hz with an Axopatch
1D patch-clamp amplifier (Axon Instruments, Foster City, CA). The
membrane potential was held at 0 mV and usually stepped to test
voltages from
100 to +80 mV in 20-mV increments at 1-s intervals (see
Figs. 7 and 11). Each step lasted 819.2 ms with intervening periods of
180.8 ms at the holding potential. A briefer voltage protocol was also
used (3), stepping the membrane potential to a smaller
number of values (
20, ± 40, and ± 80 mV).
Results are presented in three formats, namely, the short-term time
dependence of whole cell currents during the course of the voltage
steps, the long-term time course of the currents before, during, and
after experimental perturbations, and the current-voltage relationships
(I-V). The short-term time traces (see Figs. 7 and 11) are
raw currents obtained during a stable recording interval associated
with a given solution. The data points of the long-term time courses
(see Figs. 1-4, 8, 9A, and 10A) are the
means of five consecutive samples within a given voltage step, obtained
immediately after the decay of any uncompensated capacitative transient
(from 17.6 to 25.6 ms after onset of the voltage step). The
I-V of Fig. 9B and Fig. 10B were
generated by averaging the results of 3-15 experiments. The error
bars present 1 SE of the mean.
The quantification of results was complicated by the frequent steady
increase in currents throughout the experiments, and several methods
were used to obtain values. The stimulation attributed to IB-MECA was
determined by subtracting the current values just before addition of
IB-MECA from values at a plateau, or the current before application of
blockers, because these drugs were not always reversible. For example,
in Fig. 1, the plateau before
application of 100 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB) was taken as the maximum current in IB-MECA. This method is
clearly dependent on the time following application of IB-MECA, and
this variability is reflected in the SE (see RESULTS). The
slow, steady increase made the concentration-response experiments
particularly difficult to analyze (Fig.
2). For these experiments, the rate of
increase obtained using one concentration was extrapolated, and the
response due to the next concentration was defined as the difference
between the extrapolated value, due to the low concentration, and the
observed value was attributed to the new, higher concentration. The
selectivity measurements were more precise because they involved
currents obtained at approximately the same time; to control for rising
current, the mean value before and after Cl
substitution
was obtained, and the difference currents were produced by subtracting
the currents 4-5 cycles after application of the Cl
-substituted solution from this mean value (see Fig.
9A). In the case of the blockers NPPB and niflumic acid,
only the current immediately preceding drug application was used to
control for the potential partial irreversibility of these drugs.

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Fig. 1.
Time course of effects of
1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl- -D-ribofuranuronamide
(IB-MECA) on whole cell currents. Each data point represents a mean of
15 current measurements at 4-ms intervals, beginning 840 ms after the
onset of the voltage step. Positive outward current is presented
upwards. The command voltages were ±80 and ±40 mV, with a holding
potential of 0 mV. The cell was perfused with a NaCl-Ringer solution
(Table 1) except for the transient perfusion with a low
Cl -containing (L) solution (NaAsp, Table 1). Currents
were activated by 100 nM IB-MECA after a lag time of ~2 min. Outward
currents were reduced by lowering the external Cl
concentration, and outward and inward currents were reversibly
inhibited by either 10 µM (n) or 100 µM (N)
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). For purposes of
clarity in this initial figure, only a subset of the 10 voltage steps
has been included.
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Fig. 2.
Concentration dependence of IB-MECA-stimulated currents. Current
was unaffected by 10 nM IB-MECA, and 30 nM produced only a small
effect, whereas a clear increase followed application of 100 nM
IB-MECA. When added after current activation, 100 nM MRS-1523 + 100 nM IB-MECA did not reduce current levels, although currents
increased at a faster rate once the MRS-1523 was removed. Current was
clearly inhibited by both 500 µM niflumic acid and 100 µM NPPB.
|
|
Volumetric measurements and analysis.
After a single T-75 flask was harvested by trypsinization
(36), a 0.5-ml aliquot of the cell suspension in DMEM (or
in Cl
-free medium, where appropriate) was added to 20 ml
of each test solution. The standard test solution contained (in mM) 110 NaCl, 15 HEPES, 2.5 CaCl2, 1.2 MgCl2, 4.7 KCl,
1.2 KH2PO4, 30 NaHCO3, and 10 glucose, at a pH of 7.4 and osmolality of 298-305
mosmol/kgH2O. The Cl
-free solution comprised
(in mM) 110 sodium methanesulfonate, 15 HEPES, 2.5 calcium
methanesulfonate, 1.2 MgSO4, 4.7 potassium methanesulfonate, 1.2 KH2PO4, 30 NaHCO3, and 10 glucose, at a pH of 7.4 and osmolality of
294-304 mosmol/kgH2O. Parallel aliquots of cells were
studied on the same day. One aliquot usually served as a control, and
the others were exposed to different experimental conditions at the
time of suspension. The same amount of solvent vehicle
(dimethylformamide) was always added to the control and experimental
aliquots. The sequence of studying the suspensions was varied to
preclude systematic time-dependent artifacts (7).
Cell volumes of isosmotic suspensions were measured with a Coulter
Counter (model ZBI-Channelyzer II), using a 100-µm aperture. As
previously described [Yantorno et al. (37)], the cell
volume (Vc) of the suspension was taken as the peak of the
distribution function. Cell shrinkage was fit as a function of time
(t) to the simple exponential function
|
(1)
|
where V
is the steady-state cell volume,
V0 is cell volume at t = 0, and
is the
time constant of the shrinkage. For purposes of data reduction, the
data were normalized to the first time point, taken to be 100%
isotonic volume. The baseline isotonic value is ~2,000 fl.
Fits were obtained by nonlinear least-squares regression analysis,
permitting both V
and
to be variables (3).
Chemicals.
All chemicals were reagent grade. IB-MECA and Cl-IB-MECA were obtained
from Research Biochemicals International (Natick, MA), NPPB was
purchased from Biomol Research Laboratories (Plymouth Meeting, PA),
niflumic acid was purchased from Sigma (St. Louis, MO), and MRS-1523
was a gift from Dr. Kenneth Jacobson (National Institutes of Health).
Statistics.
Values are presented as the means ± 1 SE. The symbol n
indicates the number of experiments, except in Fig. 1, where it
represents the addition of 100 µM NPPB. The probability
(P) of the null hypothesis was tested with Student's
two-tailed t-test.
 |
RESULTS |
Stimulation of A3 receptor.
The selective A3 receptor agonist IB-MECA (100nM)
(14, 17) activated whole cell currents in
human NPE cells (Fig. 1). An increase in current was initially detected
1-4 min after application of IB-MECA, and we have estimated that
IB-MECA induced an 88 ± 31% rise in currents over baseline
(n = 13). Because the current rose continually in some
experiments, and in these cases the measurement of maximum current is
time dependent, this is only a rough measurement (see
METHODS). However, the value (and its variability) is
comparable to the increases produced by a 100-fold higher concentration
of adenosine (199 ± 54%; n = 4) and the
nonmetabolizable analog 2-chloroadenosine (136 ± 45%;
n = 10) (3).
Previous studies have shown that adenosine reduced cell volume in these
NPE cells primarily by activating the A3-receptor subtype
(22). Because IB-MECA is 50-fold more selective for A3 receptors than for A1 or A2a
receptors (14, 16, 17), the
ability of IB-MECA to stimulate currents in the present study suggests
that A3 receptors are also involved and that the volume response and current stimulation may share a common mechanism. Several
experiments were performed to determine the specific role of the
A3 receptor and whether the volume response and current stimulation are linked. First, the concentration dependence of the
current activation was examined; 10 nM IB-MECA did not produce a
significant increase in current (n = 3), 30 nM produced
a small, 19 ± 9% rise in current (n = 3), and in
two experiments, 1 µM IB-MECA did not produce any larger response
than 100 nM (Fig. 2). This is similar to the concentration-dependent
action of IB-MECA on cell volume, where the dissociation constant
(Kd) was 55 nM (22). Next,
the specificity of the current was examined using the highly selective
A3 agonist Cl-IB-MECA, which has an inhibitory constant
(Ki) for A3 receptors >1,400 times
lower than for A1 or A2a receptors
(16). Cl-IB-MECA clearly activated currents in duplicate
experiments (Fig. 3), and the ability of
10 nM to induce a response is consistent with the fact that Cl-IB-MECA has a lower Ki at the A3 receptor
than IB-MECA. The specificity of the response for the A3
receptor was further shown with MRS-1523, which antagonizes
A1, A2, and A3 receptors with a
Ki (in nM) of 15,600, 2,050, and 19, respectively (18). In two experiments, MRS-1523 prevented
activation of whole cell currents by IB-MECA when the two substances
were presented simultaneously. The minimal inhibition observed when
MRS-1523 was presented after the response to IB-MECA was underway (Fig.
2) is consistent with its block at the receptor level. Occasionally,
the response desensitized, and increasing the concentration of
A3 agonist (in this case adenosine) did not produce
additional increase in current (Fig.
4A). The response was clearly
uncontaminated by P2 ATP receptor contribution, because
addition of ATP induced an increased response of inward currents even
when the A3 receptor was desensitized (Fig. 4B).

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Fig. 3.
Activation of current by Cl-IB-MECA. The rate of increase was
increased by 30 nM and 100 nM Cl-IB-MECA, but addition of 1 µM
Cl-IB-MECA produced no further increase. Current was blocked by both
500 µM niflumic acid (Nif) and 100 µM NPPB.
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Fig. 4.
Response does not involve ATP receptor. A: the
response occasionally desensitized, and application of the
A3 agonist adenosine (Aden), even at higher concentrations,
did not elicit a response. Iso, isotonic central solution.
B: even after the A3 receptors had desensitized
to 1 mM adenosine, addition of 1 mM ATP activated currents at
hyperpolarized potentials, indicating that different receptors were
involved. This trace is a continuation of the experiment in
A. The decrease in current at depolarized potentials
probably represents the block of outward pICln
Cl currents by ATP.
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Role of Cl
channels.
The reduction in cell volume by A3-receptor stimulation was
found to involve the movement of Cl
from the NPE cells
(22), so we asked whether the current activated by IB-MECA
was carried by Cl
ions. Several observations suggest that
the response does involve Cl
channels. Lowering the
external Cl
concentration from 140 (NaR solution) to 25 mM (NaAsp solution) by equimolar aspartate substitution (Table 1)
dramatically reduced the outward currents during IB-MECA perfusion but
had little effect on the inward whole cell currents (Fig. 1). This is
consistent with the movement of Cl
ions because the
outward currents largely reflect entry of Cl
from the
bath, whereas inward currents largely reflect outward movement of the
intracellular Cl
at a fixed concentration throughout the experiment.
The pharmacological profile also implicates the activation of
Cl
channels. At 100 µM, the Cl
channel
blocker NPPB nearly abolished both outward and inward currents
activated by IB-MECA (Figs. 1 and 2) and by Cl-IB-MECA (Fig. 3). NPPB
is known to block K+ as well as Cl
channels
(26), but K+ was omitted from the internal and
external solutions for just this reason (Tables 1 and 2). At a
concentration of 10 µM, NPPB is thought to be free of nonspecific
effects (32). As displayed in Fig. 1, NPPB exerted
qualitatively similar effects at 10 and 100 µM. Niflumic acid, which
also blocks Cl
channels (33), showed a
similar inhibition when applied to IB-MECA-activated (Fig. 2) and
Cl-IB-MECA-activated currents (Fig. 3) at a concentration of 500 µM.
To further strengthen the link between the reduction in cell volume and
the activation of Cl
channels by A3-receptor
stimulation, the effect of NPPB on cell shrinkage was tested. IB-MECA
(100 nM) led to a reduction in cell volume that was inhibited by 100 µM NPPB (Fig. 5).

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Fig. 5.
Block of cell shrinkage by NPPB. IB-MECA (100 nM) led to
a reduction in the volume of nonpigmented ciliary epithelial cells
(V = 95.1 ± 0.3, = 2.9 ± 0.7 min) that was blocked by 100 µM NPPB. The block appeared to be
delayed, taking effect only 4 min after simultaneous presentation of
both IB-MECA and NPPB.
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|
As illustrated by Fig. 6, the reversal
potential (Erev) for the IB-MECA-activated
currents was well estimated by nonlinear least-squares fit (SigmaPlot)
to a form of the Goldman equation and was always used to calculate the
reversal potentials
|
(2)
|
where
|
(3)
|
|
(4)
|
and Vm is the membrane potential,
PCl is the Cl
permeability,
F is the Faraday's constant,
[Cl
]o is the external Cl
concentration, R is the perfect gas constant, and
T is the absolute temperature. Estimates for both unknown
parameters (PCl and Erev) were generated by the analysis. With the use of this approach, the
reversal potential (corrected for junction potential) for the
IB-MECA-activated currents in the NaCl-Ringer solution was
27.0 ± 1.9 mV (n = 13). No ion other than Cl
could account for this value of reversal potential (Table 1). The NPPB
difference currents, obtained by subtracting values during the NPPB
block from the IB-MECA-stimulated values immediately proceeding drug
application, displayed a corrected reversal potential of
30.2 ± 2.7 mV. These estimates of reversal potential are less than the
theoretical Nernst potential for Cl
(
43.5 mV) and could
reflect the contribution of aspartate movement out of the micropipette.
As discussed below, the differences from ideal Nernst behavior could be
accommodated by a relative permeability for aspartate to
Cl
(PAsp/PCl) of
0.18-0.23.

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Fig. 6.
Goldman fit of the IB-MECA-activated difference currents
observed in NaCl-Ringer solution (Table 1). The figure presents a
representative example (continuous trajectory) of the fits generated
with the Goldman equation. Nonlinear least-squares fit to Eq. 1 generated values for PCl and
Vm of 13.5 ± 0.5 fl/s and 22.9 ± 1.9 mV, respectively. From the mean cell capacitance of this series
(32.4 ± 6.2 pF) and taking the capacitance/area ratio to be 1 µF/cm2, the value generated for
PCl can also be expressed as 4.2 ± 0.8 × 10 7 cm/s.
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Figure 7 presents the time courses of
difference currents after step changes in voltage. The
IB-MECA-stimulated difference currents showed similar outward
rectification in both high- (Fig. 7A) and
low-Cl
(Fig. 7, C and D) containing
solutions, and inactivation at positive potentials. A similar pattern
was displayed for the currents blocked by NPPB in high-Cl
solution (Fig. 7B). Comparable outward rectification and
inactivation was seen in the NPPB-inhibited currents (Fig.
7B) and in currents activated by nonspecific
adenosine-receptor agonists (Fig. 7; Ref. 3).

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Fig. 7.
IB-MECA and NPPB difference currents as functions of time following
voltage steps. The IB-MECA difference currents were generated by
subtracting the currents in the control period from the corresponding
currents during the response to the A3 agonist in NaCl
(A), NaAsp (C), and NaCycl (D) (see
Table 1). The NPPB difference currents were measured as the
NPPB-sensitive currents (B) in the presence of 100 nM
IB-MECA in NaCl-Ringer solution. Here and in Fig. 11, the traces
represent currents obtained from 100 mV through +80 mV in 20-mV
steps, with the top trace in each figure showing the +80 mV
trace.
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Effects of ionic composition on response to IB-MECA.
One of the identifying characteristics of Cl
channels is
their anionic selectivity. For example, Voets et al. (30)
differentiated between one channel associated with injection of message
for the human volume-sensitive Cl
conductance regulatory
protein pICln and another endogenous volume-activated channel, in part by a striking difference in the relative
permeabilities to Cl
, NO3
, and
cyclamate. Human pICln was first cloned in the cell line currently studied (1), and these cells also display
swelling-activated Cl
channels (36). We
have, therefore, examined the relative permeabilities to
Cl
, NO3
, aspartate, and cyclamate, both
of IB-MECA-activated and swelling-activated Cl
channels
in these cultured human NPE cells.
Before the relative anionic permeability was calculated, the cation
selectivity of the IB-MECA-stimulated current was determined by
alternately perfusing the cell with the NaCl, NaAsp, NMDG-Cl, and
NMDG-Asp Ringer solutions of Table 1. Figure
8 demonstrates that the effect of
substituting NMDG for Na+ was very small before and after
IB-MECA was added. The striking observation was the dependence of the
whole cell currents on external Cl
. The corrected
reversal potential for IB-MECA-activated channels in the presence of
NMDG-Cl solution (Table 1) was
28.4 mV, almost identical with that in
NaCl-Ringer solution (
27.0 mV, Table
3). Thus the IB-MECA-activated channels
cannot distinguish between Na+ and NMDG in contrast to
their discrimination between Cl
and aspartate.

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Fig. 8.
Time course of effects of IB-MECA as a function of dominant
external cation. Each data point represents a mean of 5 current
measurements at 1.6-ms intervals, beginning 17.6 ms after the onset of
the voltage step. The holding potential was 0 mV, and command voltages
were stepped from +80 to 100 mV in 20-mV decrements. During the
control period, substitution of
N-methyl-D-glucamine (NMDG)+ for
Na+ had little effect on the whole cell currents, but
reduction of the external Cl by aspartate (Asp)
replacement markedly lowered the outward currents. The same
observations were noted after stimulation with IB-MECA, and all
currents were markedly inhibited by 100 µM NPPB added at the
conclusion of the experiment.
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The anion selectivity was explored by perfusing the same cell
with the Na+ salt of different anions (Fig.
9A). Both before and during
perfusion with IB-MECA, partial Cl
replacement with
aspartate reduced the outward positive currents. Cyclamate reduced the
outward currents even more, and in addition lowered the inward currents
(more easily appreciated in Fig. 9C). The application of
NPPB nearly abolished inward and outward currents. Figure 9,
B and C, presents the averaged reduced data in
the form of I-V relationships. The IB-MECA-activated
difference currents were similar in NaCl- and NaNO3-Ringer
solutions, and both were also similar to the NPPB difference currents
measured in the presence of IB-MECA in NaCl-Ringer solution (Fig.
9B). The corrected reversal potentials (Table 3) for these
difference currents were
27 mV (NaCl),
27.6 mV (NaNO3),
and
30.2 mV (NPPB in NaCl).

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Fig. 9.
Effects of IB-MECA as a function of the dominant external anions.
A: perfusion with 100 nM IB-MECA increased the total
currents and Cl -dependent fractions of those currents.
Partial replacement of Cl with cyclamate (Cycl) (but not
aspartate) produced a small but clear inhibition of inward current only
after activation by IB-MECA. NPPB produced a nearly complete,
reversible inhibition of inward and outward currents. The
Na+ form of the salts was used. The voltage protocol and
analysis procedure of Fig. 7 were applied. B: difference
currents for cells perfused with NaCl- and NaNO3-Ringer
solutions, and NPPB difference currents for stimulated cells following
IB-MECA activation. C: difference currents for cells
perfused with NaAsp- and NaCycl-Ringer solutions for cells activated by
IB-MECA.
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Approximate values of the relative permeabilities can be calculated for
aspartate (PAsp/PCl) and
nitrate (PNO3/PCl) from
the reversal potentials of Table 3, using the Goldman equation
|
(5)
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(6)
|
The calculated values for
(PAsp/PCl) and
(PNO3/PCl) through
the IB-MECA-activated channels are 0.23 and 1.03, respectively.
NaAsp-Ringer solution reduced the outward currents and shifted the
corrected reversal potential to
3.1 mV. The inward currents were also
slightly reduced during the course of NaAsp perfusion (compare Fig. 9,
B and C). This reduction in inward currents was much more marked during partial cyclamate substitution for
Cl
(Fig. 9C). The cyclamate replacement for
Cl
also markedly reduced the outward currents. The
possible differential effects of cyclamate and aspartate on the inward
currents were examined by attempting to scale the difference currents
in NaAsp (
INaAsp) and NaCycl
(
INaCycl) to those in NaCl
(
INaCl) with the scaling factors
kAsp and kCycl,
respectively
|
(7)
|
|
(8)
|
Over the depolarizing voltage range
(Vm > 0 mV),
INaCl was well fit by Eq. 7 with
kCycl = 11.8 ± 0.3, and with
kCycl = 10.7 ± 0.9 over the
hyperpolarizing domain. The difference between these values of the
scaling factor (1.1 ± 0.9) was not statistically significant. In
contrast, the values generated for kAsp with
Eq. 8 over the hyper- and depolarizing ranges differed by
0.7 ± 0.2 (P < 0.02). These calculations
indicated that external cyclamate produced a voltage-independent
reduction in the IB-MECA-activated difference currents, whereas
substitution of aspartate for Cl
had a greater effect on
the outward than on the inward difference currents. The reversal
potential in NaCycl-Ringer solution was estimated to be
32.5 mV, but
this value is subject to considerable uncertainty given the very small
values of the recorded difference currents.
Effects of ionic composition on swelling-activated Cl
channels.
We used a protocol similar to that of Fig. 9A in examining
the effects of external Cl
, NO3
,
aspartate, and cyclamate on currents through swelling-activated Cl
channels in the same line of human NPE cells. As
illustrated by Fig. 10A, the
effects of partial replacement of external Cl
by
aspartate or cyclamate were qualitatively similar to those noted for
the IB-MECA-activated channels (Fig. 9A). Aspartate appeared
to reduce the outward currents with little effect on the inward
currents, whereas cyclamate reduced both outward and inward currents
equally. Once again, this differential effect was suggested by using
Eqs. 7 and 8 in estimating the scaling factors
mapping the difference currents in NaAsp and NaCycl to those in NaCl
(Table 2). The scaling factor for cyclamate was not significantly
different in the hyper and depolarizing voltage ranges
[(
kCycl)Swell = 0.2 ± 0.1], whereas the difference in scaling factors for aspartate was
significant [(
kAsp )Swell = 0.27 ± 0.06; P < 0.01]. NPPB markedly inhibited
both inward and outward currents.

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|
Fig. 10.
Effects of
swelling as a function of the dominant external anions. A:
the swelling-activated currents displayed a similar dependence on the
dominant external anion. Partial substitution of external
Cl with I was noted to have only a small
effect (as seen here) and was not therefore systematically pursued in
this study. The mean swelling-activated currents of 3 experiments were
similar with NaCl- and NaI-Ringer solutions, but the corrected reversal
potential was shifted negatively by 2.5 ± 0.9 mV by the
I substitution. B: difference currents for
cells perfused with NaCl- and NaNO3-Ringer solutions, and
NPPB difference currents for stimulated cells after cell swelling.
C: difference currents for cells perfused with NaAsp- and
NaCycl-Ringer solutions for cells activated by hypotonic
perfusion.
|
|
The mean swelling-activated difference currents measured in the
various perfusates are displayed in Fig. 10, B and
C. As in the case of the IB-MECA-activated currents, the
I-V relationships in NaCl- and NaNO3-Ringer
solutions were similar to each other and to the NPPB difference
currents obtained in NaCl-Ringer solutions. Once again, aspartate
substitution for Cl
reduced the outward currents and
shifted the reversal potential. The magnitudes of all the activated
currents were larger in this series of experiments than those of the
IB-MECA-activated currents of Fig. 9. The effects produced by partial
replacement of external Cl
with cyclamate can therefore
be more readily identified. As noted above, there was a marked
reduction in both the outward and inward currents. In the case of Fig.
10C, the reversal potential in the NaCycl-Ringer solution
can now be more clearly identified and is not significantly different
from that of aspartate.
We used equations analogous to Eqs. 5 and 6 to
estimate the relative permeabilities for Cl
, aspartate,
and NO3
through the swelling-activated channels. With
the use of the corrected reversal potentials entered in Table
4, we calculated values of 0.21 for
(PAsp /PCl) and 0.87 for (PNO3/PCl).
Figure 11 presents the time courses for
representative difference currents in response to voltage pulses across
the swelling-activated channels. Both the swelling-activated difference
currents in different anions and the NPPB difference current in
NaCl-Ringer solution are very similar to the corresponding traces for
the IB-MECA-activated channels (Fig. 7). A prominent inactivation was
noted at highly depolarizing voltages, and a variable small activation
was noted at highly hyperpolarizing potentials.

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|
Fig. 11.
Swelling-activated and NPPB difference currents as functions of
time following voltage steps. As in Fig. 6, swelling-activated
difference currents were generated by subtracting the currents in the
control period from the corresponding currents during the response to
hypotonic perfusion with solution of the same ionic composition [NaCl
(A), NaAsp (C), and NaCycl (D); see
Table 2]. The NPPB difference currents (B) were measured as
the NPPB-sensitive currents in hypotonic NaCl-Ringer
solution.
|
|
 |
DISCUSSION |
The major findings of the present study are that the selective
A3-subtype adenosine agonist IB-MECA activates
Cl
channels and that the anionic dependence of these
channels is similar to that of the swelling-activated Cl
channels of the same human line of nonpigmented ciliary epithelial cells.
We have previously reported that adenosine and nonspecific analogs
activate Cl
channels of cultured human NPE cells
(3), consistent with reports that A1-subtype
adenosine receptors activate a 305-pS Cl
channel in
RCCT-28A rabbit cortical collecting duct cells (29) and
that Cl
channels are likely involved in
adenosine-mediated effects in rat hippocampal slices (27).
However, a previous report implicating A3-subtype adenosine
receptors in the modulation of Cl
channels was based on
indirect evidence from measurements of cell volume, short-circuit
current, and RT-PCR amplification of RNA from cells and tissue
(22). Use of highly selective agonists and antagonists
permitted unambiguous demonstration that the observed effects of
adenosine were mediated through A3 receptors, but the evidence that Cl
channels were indeed activated was less
compelling. In the present study, we have observed that the
A3-selective agonist IB-MECA activates Cl
channels with a dose dependence similar to the volume response, that
similar currents are stimulated by the highly-selective agonist Cl-IB-MECA, that the reversal potential for the IB-MECA-stimulated currents is strongly dependent on the external Cl
concentration, that the currents can be inhibited by niflumic acid (500 µM) or by a low concentration (10 µM) of the Cl
channel blocker NPPB and essentially abolished by a higher
concentration (100 µM), and that this higher concentration of NPPB
also blocks the shrinkage induced by IB-MECA. It seems reasonable to
conclude that A3-receptor specificity (established in Ref.
22) is mediated by activation of Cl
channels (documented
in the current work).
From measurements of the reversal potential, we have calculated that
the relative permeabilities through the IB-MECA-activated channels are
PNO3 ~ PCl
PAsp. We cannot estimate the relative permeability for cyclamate because its application to the external solution blocks both inward and outward currents by a mechanism yet to
be identified. Perfusion with external cyclamate reduced the inward
current conductance of both the IB-MECA- and swelling-activated currents by at least threefold (P < 0.001). This
effect appears to be voltage independent (Fig. 9C) and
could, therefore, reflect slow permeation through the pore or blocking
action outside the conduit.
Single-channel patch-clamp measurements of bovine nonpigmented ciliary
epithelial cells have identified at least two types of
swelling-activated Cl
channels (38), and (in
principle) other functionally heterogeneous Cl
channels
could also be present. We had anticipated that perfusion with different
anions [especially cyclamate (30)] might permit us to
distinguish functionally between the IB-MECA-activated and swelling-activated Cl
channels. However, the relative
permeabilities for Cl
, NO3
, and
aspartate were similar for IB-MECA-activated and swelling-activated Cl
channels, and cyclamate produced a similar block in
each. Small differences were indeed detected. For example, the relative
permeability for NO3
was 1.03 for the
adenosine-activated and 0.86 for the swelling-activated channels. However, these small differences can reflect the
uncertainties of the techniques, including the necessarily approximate
corrections for junction potential. The measured junction potentials
agreed with the values calculated with the Henderson equation within 2.4 mV. If we take this value to represent the uncertainty of the
reversal potential measurement, we calculate that a channel with a true
PNO3/PCl ratio of 1 could
display apparent values of 0.89 to 1.12, accounting for the range of
values currently calculated.
We conclude that 1) the stimulation of A3
receptors by IB-MECA activates Cl
channels, and
2) IB-MECA-activated and swelling-activated Cl
channels of cultured human NPE cells are functionally similar in terms
of their dependence on external Cl
,
NO3
, aspartate, and cyclamate concentration, their
inhibition by NPPB, and their inactivating characteristics following
step changes in voltage. Previous studies on the short-circuit current
across rabbit iris ciliary body have suggested that the
Cl
conductance activated by adenosine (3)
and IB-MECA (22) is present on the basolateral surface of
the NPE cells facing the aqueous humor. Activation of these channels
would thus permit efflux of Cl
out of the NPE cells and
into the interior of the eye and a corresponding increase in the rate
of aqueous humor production. The similarity between the
IB-MECA-stimulated and the swelling-stimulated Cl
currents shown in the present study suggests that the latter could also
be linked to an increase in aqueous humor production. We hypothesize
that adenosine arises from the extracellular breakdown of ATP released
from pigmented ciliary epithelial and NPE cells (21) and
that substances that lead to ATP release, for example, those which
elevate Ca2+, could lead to the stimulation of
A3 receptors. The physiological trigger capable of
activating the swelling-associated pathway remains to be elucidated.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Kenneth Jacobson (National Institutes of
Health) for the MRS-1523. The compound Cl-IB-MECA (MH-C-7-08; Lot No.
CM-VIII-12) was provided by Research Biochemicals International as part
of the Chemical Synthesis Program of the National Institute of Mental
Health, Contract N01MH30003.
 |
FOOTNOTES |
Supported in part by National Eye Institute Research Grants EY-08343,
EY-11213, and EY-01583 (for core facilities) and National Heart, Lung,
and Blood Institute Respiratory Training Grant HL-07027 (to C. H. Mitchell).
Address for reprint requests and other correspondence: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, A303 Richards Bldg., 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. §1734 solely to indicate this fact.
Received 17 November 1999; accepted in final form 7 March 2000.
 |
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