Departments of 1 Physiology and 3 Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085; 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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Ciliary
epithelial cells possess multiple purinergic receptors, and occupancy
of A1 and
A2 adenosine receptors is
associated with opposing effects on intraocular pressure. Aqueous
adenosine produced increases in short-circuit current across rabbit
ciliary epithelium, blocked by removing
Cl and enhanced by aqueous
Ba2+. Adenosine's actions were
further studied with nonpigmented ciliary epithelial (NPE) cells from
continuous human HCE and ODM lines and freshly dissected bovine cells.
With gramicidin present, adenosine (
3 µM) triggered isosmotic
shrinkage of the human NPE cells, which was inhibited by the
Cl
channel blockers
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 µM, the nonmetabolizable analog 2-chloroadenosine and AMP also
produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine
(
1 µM) triggered increases of whole cell currents in HCE cells,
which were partially reversible,
Cl
dependent, and
reversibly inhibited by NPPB. Adenosine (
10 µM) also stimulated
whole cell currents in bovine NPE cells. We conclude that occupancy of
adenosine receptors stimulates
Cl
secretion in mammalian
NPE cells.
cell volume; whole cell recording; short-circuit current; 2-chloroadenosine; adenosine 5'-triphosphate; purines
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INTRODUCTION |
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THE CILIARY EPITHELIUM secretes the aqueous humor from the underlying stroma into the posterior chamber of the eye (10). This epithelium is structurally unique in consisting of two layers of cells whose apical faces are apposed. The basolateral surfaces of the pigmented ciliary epithelial and nonpigmented ciliary epithelial (NPE) cells abut the stroma and aqueous humor, respectively. The cells within each layer and between the two layers intercommunicate through gap junctions (9, 20), forming a functional syncytium. After uptake at the stromal-syncytial interface, ions diffuse through the gap junctions and are released across the basolateral membranes into the aqueous humor. The small negative transepithelial potential provides a driving force for Na+ transfer from the stroma into the aqueous humor through the paracellular pathway in parallel with the transcellular pathway.
The circadian rhythm of aqueous humor formation is the most striking indication that this secretion is regulated. The rate of secretion in humans has been reported to be two- to threefold higher during the day than during the early morning hours (1). The basis for the phenomenon remains unknown, although attention has been addressed to the potential roles of many hormones and of sympathetic nerve activity (1). Among the mediators considered to be possible triggers of circadian rhythms has been adenosine (5). The potential role of purinergic agonists is of particular interest, since activation of adenosine A2 receptors has been associated with ocular hypertension (12), while occupancy of adenosine A1 receptors has been associated with ocular hypotension (11).
Despite the potential importance of purinergic regulation of aqueous humor formation, very little information is available concerning the effects of adenosine on isolated tissue preparations. Adenosine increases the adenosine 3',5'-cyclic monophosphate (cAMP) level of the rabbit iris-ciliary body (11) and acts synergistically with acetylcholine to increase the Ca2+ activity of NPE cells in rabbit ciliary processes (14). The purpose of the present study was to determine whether adenosine exerts effects on transport by the ciliary epithelial cells. For this purpose, we have studied the intact rabbit iris-ciliary body, immortalized lines of human NPE cells, and freshly dissected bovine NPE cells. The immortalized human NPE cells are known to possess several forms of adenosine and ATP receptors (23, 24).
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MATERIALS AND METHODS |
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Transepithelial measurements. Adult male Dutch belted rabbits weighing 1.8-2.4 kg (Ace Animals, Boyertown, PA) were anesthetized with pentobarbital and killed (2). Treatment and death were in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. After enucleation, the iris-ciliary body was isolated, as previously described (2).
The pupil and central area between pupil and ciliary processes were
occluded with a Lucite disk, and the iris-ciliary body was mounted
between the two halves of a Lucite chamber (2). The annulus of exposed
tissue was bounded by outer and inner diameters of 12 and 5 mm,
respectively, exposing a projected surface area of 0.93 cm2. Preparations were bathed with
a Ringer solution (T1; Table 1) that was continuously bubbled with 95%
O2-5%
CO2 for maintenance of pH 7.4. Where appropriate, tissues were also bathed with a Cl-free Ringer solution
(T2; Table 1). The transepithelial potential was fixed at 0 mV and corrected for solution series resistance, and the short-circuit
current (Isc)
was monitored on a chart recorder.
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Cellular models. The ODM (17) and HCE immortalized NPE cell lines were developed by M. Coca-Prados from primary cultures of human epithelium. Cells were grown in Dulbecco's modified Eagle's medium (DMEM; no. 11965-027, GIBCO BRL, Grand Island, NY; and no. 51-43150, JRH Biosciences, Lenexa, KS) with 10% fetal bovine serum (A-1115-L; HyClone Laboratories, Logan, UT) and 50 µg/ml gentamicin (no. 15750-011; GIBCO BRL) at 37°C in 5% CO2 (26). The medium had an osmolality of 328 mosmol/kgH2O. Cells were passaged every 6-7 days and, after reaching confluence, were studied within 6-10 days after passage.
NPE cells were also obtained from freshly dissected bovine ciliary processes by the method of Jacob (16). The tips of the ciliary processes were excised and washed three times in a small volume of Ca2+- and Mg2+-free phosphate-buffered saline (PBS) solution. The tips were then transferred to 20 ml of a PBS solution containing 0.25% trypsin and 0.02% EDTA and digested at 37°C for 20-30 min with agitation. Thereafter, 10 ml of DMEM containing 10% fetal calf serum and gentamicin (100 µg/ml) were added to halt enzyme activity, and the residual tips were discarded. The preparation was next triturated to further dissociate the single cells and was centrifuged. The pellet was resuspended in PBS and spun again to remove blood cells and detritus. The cells were occasionally studied within 4 h of isolation. More commonly, the pellets were resuspended in medium containing 10% fetal calf serum, added to coverslips, and incubated overnight.
Measurement of cell volume. After harvesting of a single T-75 flask by a widely used trypsinization approach (26), a 0.5-ml aliquot of the cell suspension (usually in saline solution V; Table 1) was added to 20 ml of the test solution. The isosmotic solution V (Table 1) was used at a pH of 7.4. Four aliquots of cells were studied on the same day. One aliquot usually served as a control, with the other three aliquots being exposed to different experimental conditions. Comparisons were drawn only between experimental and control aliquots harvested from the same flask on the same day. The sequence of studying the suspensions was always varied to preclude systematic time-dependent artifacts. The stability of the control measurements was found to be enhanced by preincubating all cells for ~30 min in the control suspending solution rather than directly transferring the cells from DMEM to the test solutions.
Cell volumes of isosmotic suspensions were measured with a Coulter counter (model ZBI-Channelyzer II), using a 100-µm aperture (7). As previously described (26), the cell volume (vc) of the suspension was taken as the peak of the distribution function. The time course of cell shrinkage in the test solution was fit with the monoexponential function
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(1) |
Ruptured-patch whole cell recording.
Cells harvested by trypsinization were resuspended and permitted to
settle and attach either to glass coverslips, which were then mounted
in a perfusion chamber, or to the bases of plastic petri dishes.
External bath (EB) solutions were adjusted to pH 7.4 (Table
2). Micropipettes were double pulled from
Corning no. 7052 glass, coated with Sylgard, and were fire polished.
The micropipette (MP) filling solutions used were
MP1,
MP2, or
MP3 at pH 7.2 or 7.4, as indicated in
Table 2. Sufficient CaCl2 was
included to generate a free Ca2+
concentration of either 10 or 100 nM. With these solutions,
micropipettes displayed resistances of 1.5-4.0 M. The seals
formed commonly had resistances of ~10-20 G
. After rupturing
the membrane patch, the series resistance was measured to be 10 ± 2 M
(n = 10).
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Data were acquired at 2 kHz using Axopatch-1B electronics and
associated headstage (Axon Instruments, Foster City, CA).
In the experiments conducted with cultured human NPE cells (see Figs. 5-8), series resistance was compensated by 80%. In a series of experiments conducted with freshly dissected bovine NPE cells, series
resistance compensation was not used, and the reduced current-voltage (I-V) relationships were corrected a
posteriori for the calculated voltage drops across the series
resistances.
The time courses of the whole cell currents (see Figs. 5 and 6) are
presented as the raw measurements. The reduced
I-V relations (see Figs. 7 and 8) have
been corrected (19) for the junction potentials measured with reference
to a flowing 3 M KCl junction, whose values were (in mV, filling
solution with respect to perfusate) 1.50 ± 0.08 (MP1/EB1H),
3.43 ± 0.05 (MP2/EB1H),
1.64 ± 0.02 (MP2/EB2H),
6.69 ± 0.09 (MP1/EB1L),
3.73 ± 0.03 (MP2/EB1L),
and 6.39 ± 0.04 (MP2/EB2L).
The compositions of the pairs of solutions (MP/EB) are presented in
Table 2.
The basic voltage protocol was to hold the potential at 0 mV, with
cycles of step changes to ±40 and ±80 mV every 10 s. Each step
usually lasted 1.3 s, with intervening periods of 0.7 or 1.3 s at the
holding potential. In one-half the experiments, the protocol was
modified to also include a voltage step of 20 mV (see Figs.
5-8).
Chemicals. All chemicals were reagent grade. Niflumic acid, adenosine, 2-chloroadenosine, inosine, AMP, ATP, UTP, gramicidin, and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) were purchased from Sigma Chemical (St. Louis, MO), and staurosporine and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) were bought from Biomol Research Laboratories (Plymouth Meeting, PA).
Data presentation. Values are means ± SE, where n is the number of experiments. The probability (P) of the null hypothesis was tested with Student's two-tailed t-test.
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RESULTS |
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Transepithelial measurements. The
results of a series of eight experiments are presented in Fig.
1. Each rabbit iris-ciliary body served as
its own series control. After a period of stable recording, 100 µM
adenosine was added to the aqueous side solution bathing each tissue
and then removed by perfusion with adenosine-free solution. In one-half
of the eight experiments, tissues were next bathed in methylsulfonate
(Cl-free) solution and then
exposed to a repeat application of 100 µM adenosine. In the other
four experiments, tissues were exposed to 5 mM
BaCl2 on both surfaces (by
isosmotic substitution for NaCl) and subsequently to adenosine. The
absolute means ± SE for the baseline open-circuit transepithelial
potential and Isc
of the eight preparations were
0.96 ± 0.08 mV (with
reference to stroma) and 6.93 ± 0.96 µA, respectively.
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After a lag time of 2-3 min, adenosine produced a stimulation of
Isc that reached
a peak change of 0.29 ± 0.04 µA within 20 min and decayed very
slowly to 0.20 ± 0.03 µA by 45 min after addition of
purine. The stimulation elicited by 100 µM nucleoside was abolished
by Cl removal. In contrast,
the K+ channel blocker
Ba2+ enhanced the magnitude of the
adenosine-induced stimulation, presumably by blocking concurrent
adenosine-triggered K+ secretion.
We concluded that adenosine was probably binding to purinergic
receptors at the aqueous surface of the NPE cells, thereby triggering
effects on ion transport across the epithelium. One of these effects
was likely to have been stimulation of
Cl release through
Cl
channels of the NPE
cells, although other interpretations were possible. Given the
complexity of the intact ciliary epithelium, we pursued this hypothesis
by studying NPE cells in isolation by measuring both cell volume and
whole cell currents.
Isotonic shrinkage of human NPE cells. Figure 2, A and B, demonstrates that adenosine produced a significant graded stimulation of isotonic shrinkage. A similar protocol was followed in the experiments of both panels. The control aliquot of cells received no adenosine, and another aliquot was exposed to the maximal concentration (10 mM) of adenosine at the time of initial suspension. The intermediate concentrations were 1 and 3 µM (Fig. 2A) and 10 and 100 µM (Fig. 2B). Adenosine triggered a shrinkage at 1 µM that was not significant at the 0.5 probability level but was highly significant (P < 0.001) at all higher concentrations. The half-maximal concentration (EC50) in Fig. 2A was <3 µM. However, biological variance was detectable among experiments conducted in different weeks, largely as a function of passage number. The EC50 in Fig. 2B was ~10 µM. The solid lines are the nonlinear least-squares regression fits to Eq. 1. Details concerning the values of the fits to the monoexponentials are provided in the legends to Figs. 2-4.
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The experiments of Figs. 2, 3, and 4A
were conducted with 5 µM gramicidin included in the control and
experimental suspending solutions. Gramicidin distributes to the plasma
membrane, providing an exit port for
K+, so that
Cl channel activity is the
rate-limiting factor for release of solute (KCl) and water (6). As
expected, adenosine stimulated a similar shrinkage in the absence of
gramicidin (P < 0.001; Fig.
4B) at all concentrations applied
[100 µM (n = 4), 1 mM
(n = 4), and 10 mM
(n = 10)]. The putative role of
Cl
channels in the
isosmotic shrinkage of Fig. 2, A and
B, was supported by data obtained with
Cl
channel blockers. Both
100 µM NPPB (n = 4, P < 0.001) and 500 µM niflumic
acid (n = 4, P < 0.001) abolished the shrinkage
produced by 10 mM adenosine (data not shown).
Adenosine receptors and second messengers. Figure 3, A and B, indicates that the transport effects of adenosine were mediated by occupancy of adenosine (P1-purinergic) receptors. Consistent with the data of Fig. 2, 10 µM adenosine produced an isotonic shrinkage of the human NPE cells (n = 3; P < 0.001; Fig. 3A). In contrast, neither ATP nor UTP exerted significant effects on parallel aliquots at the same concentration, an observation incompatible with mediation through ATP (P2-purinergic) receptors (13, 15). Nevertheless, the responses to adenosine might possibly have been mediated by inosine, a deaminase product of adenosine, or by some later metabolite of inosine. This possibility was addressed by comparing the effects of 10 µM concentrations of inosine, 2-chloroadenosine (a nonmetabolizable analog of adenosine), and another purine agonist of adenosine receptors (AMP) (15) on the rate of isosmotic cell shrinkage (n = 4, Fig. 3B). Inosine had no effect, but both 2-chloroadenosine and AMP significantly accelerated shrinkage (P < 0.001), consistent with the effects of these purines on adenosine receptors in other tissues (13).
The results of Fig. 4, A and B, provide preliminary information concerning the possible second-messenger cascades involved in adenosine's actions on NPE transport. Some effects of stimulating adenosine receptors are mediated by changes in the intracellular cAMP level (13). The possible interaction of cAMP and adenosine was examined by adding 1 mM DBcAMP, 10 mM adenosine, and both agents together to parallel aliquots of cells (n = 10, Fig. 4B). Each agent alone produced shrinkage (P < 0.001), and the combined effect was at least additive (P < 0.001). The time constant was significantly smaller for cells exposed to cAMP and adenosine together than to either agonist separately (P < 0.001), suggesting that cAMP does not mediate the adenosine-triggered shrinkage.
Protein kinase C (PKC) has also been implicated in the transport
effects of adenosine. Inhibiting baseline PKC activity blocks adenosine
activation of a Cl channel
in RCCT-28A cells (22). Actually, blocking PKC activity with 0.3 µM
staurosporine stimulated shrinkage (n = 4, P < 0.005; Fig.
4A), as previously reported (6).
This shrinkage was significantly accelerated by adding 10 mM adenosine
together with the staurosporine (P < 0.001). The converse was not true. Adding the two agents together did
not further affect the rate of shrinkage triggered by 10 mM adenosine
alone (P > 0.4). Thus the actions of
adenosine cannot be mediated solely through an inhibition of endogenous PKC activity.
Whole cell patch clamping. NPE cells were also patch clamped to examine the effects of purines under controlled conditions of intracellular composition and membrane potential. Figures 5-8 present results obtained by perfusing HCE human NPE cells with 2-chloroadenosine, the nonmetabolizable analog of adenosine. At a concentration of 10 µM, 2-chloroadenosine increased the whole cell currents of each of the 10 cells examined (P < 0.001; Figs. 5-8). The increase in current at the most depolarized potential (80 mV) was 1,420 ± 371 pA, corresponding to a stimulation of 136 ± 45% over baseline current. The effect was at least partially reversible in cells after prolonged washout with fresh perfusate (~10-15 min, e.g., Fig. 5), but this point was not systematically examined. The stimulation could be repeated in those cells, using the same 10 µM concentration of adenosine (Figs. 5 and 6). Stimulation of whole cell currents was also detectable in each of four experiments at a concentration of 1 µM (Fig. 6). The measured increase was 190 ± 92 pA, a stimulation of 36 ± 21%. For purposes of comparison, 10 µM adenosine increased the current by 1,519 ± 965 pA (199 ± 54%, P < 0.05) in the same set of four experiments.
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Three observations indicated that the stimulated currents largely
reflected Cl channels.
First, purine-stimulated outward whole cell currents were strongly and
reversibly dependent on the external
Cl
concentration (Figs.
5-8). Second, the Cl
channel blocker NPPB (100 µM) produced a large reversible inhibition (57 ± 15%, n = 4, P < 0.05) of both outward and inward
currents (Figs. 5-6). NPPB is known to block
K+ as well as
Cl
channels (21), but
K+ was omitted from the
micropipette filling solution for just this reason (Table 2). Third,
the reversal potential
(Erev) of the adenosine difference currents (Figs. 7-8) was shifted to more
positive values by reducing the external
Cl
concentration.
The difference in
Erev
(Erev) in
the high and low
Cl
-containing perfusates
was less than the expected value of
37.3 mV for perfectly
Cl
-selective channels. This
issue was most readily addressed in the set of four experiments
conducted with the bicarbonate-free solutions
(MP2,
EB2H, and
EB2L; Table 2).
The ratio of the permeability of the anion (A) aspartate to that of
Cl
(PA/PCl)
through the 2-chloroadenosine-stimulated channels was estimated from the Goldman equation in the
form
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(2) |
The kinetics of the adenosine difference currents of Fig. 5 are
presented in Fig. 7. Significant inactivation was noted only at 80 mV,
with time constants of 284 ± 3 and 202 ± 3 ms in high (Fig.
7A) and low (Fig.
7B) external
Cl concentrations,
respectively. Figure 7, C and
D, presents the voltage dependence of
the adenosine-stimulated currents measured early (4-24 ms) and
late (1.17-1.19 s) after initiating each voltage step. Outward
rectification was observed with external and internal Cl
concentrations of 122.1 and 22.9 mM, respectively. The rectification was far more prominent for
the peak I-V relation, given the
inactivating component at large depolarizing potentials (Fig. 7,
A and
B).
Freshly dissociated bovine NPE cells were also patch clamped to
determine whether adenosine exerted similar electrophysiological effects on NPE cells other than simian virus 40-immortalized cell lines. In this series, the filling solution of the micropipette was
MP3 and the perfusate was
solution EB3 (Table 2), so that the
Nernst potentials were (in mV) 0 (Cl),
42
(K+), 78 (Na+), and 51 (for
cation-nonselective channels). Adenosine (10 µM to 1 mM) stimulated
whole cell currents of each of 13 cells with an
Erev equal to the
Cl
Nernst potential. In an
initial series of nine experiments, adenosine increased the current at
the most depolarized command voltage (80 mV) by 85 ± 24 pA
(P < 0.01), a stimulation of 32 ± 10% over baseline current. In view of the known coupling of
adenosine receptors to G proteins (15), GTP was included in the
micropipette filling solution in four additional experiments [10
µM (n = 1); 100 µM (n = 3)]. For the entire series
of 13 experiments, the mean increase in current at a command potential
of 80 mV was 73 ± 18 pA (P < 0.005), corresponding to a stimulation of 28 ± 7% over baseline current. The percentage stimulations at the three adenosine
concentrations applied were 12 ± 4% (10 µM,
n = 4), 28 ± 6% (100 µM,
n = 1), and 35 ± 10% (1 mM,
n = 4).
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DISCUSSION |
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Using Isc, cell volume, and whole cell currents as indexes of transport, the results demonstrate that adenosine and 2-chloroadenosine modulate the transport properties of NPE cells. Several observations indicate that the effects were triggered by occupancy of adenosine (purinergic P1 type) receptors. First, adenosine and AMP triggered isosmotic cell shrinkage, but ATP and UTP were ineffective at the same concentration (Fig. 3A), a ranking characteristic of P1 receptors and incompatible with ATP (purinergic P2 type) receptors (13, 15). Second, inosine (the deaminase product of adenosine) did not affect cell volume, whereas the nonmetabolizable analog 2-chloroadenosine stimulated both isosmotic shrinkage (Fig. 3B) and whole cell currents of HCE human NPE cells (Figs. 5-8). Third, the concentrations (1-3 µM) at which adenosine and 2-chloroadenosine stimulated shrinkage (Fig. 2A) and whole cell currents (Fig. 5) are consistent with the EC50 values at which these agonists stimulate both the A2b subtype (5-20 µM) and the A3 subtype (>1 µM) of P1 purinoceptors to alter intracellular cAMP (15). In this respect, it should be noted that Wax et al. (24) have reported that adenosine increased the cAMP content of both NPE and pigmented epithelial cells, but only at concentrations >100 µM (see p. 91 of Ref. 24). Lower concentrations have also been reported to have effects on the ciliary epithelium, but only in synergism with another agonist (14). Administered together with 10 µM acetylcholine, adenosine produced a marked synergistic increase in Ca2+ activity of rabbit ciliary processes with an EC50 of 0.25 µM. However, the change in Ca2+ activity produced by applying 1 µM adenosine alone was only 30 ± 17%, an effect of uncertain statistical significance (14).
From our results obtained with ion substitutions and channel
inhibitors, at least one of the targets of the
P1 purinergic receptors must be
Cl channels of the NPE
cells. This conclusion is based on the observations that
1) the stimulation of
Isc triggered by
aqueous adenosine was entirely abolished by substituting
methylsulfonate for Cl
(Fig. 1); 2) adenosine produced
shrinkage of cultured human NPE cells, even when release of
intracellular K+ was not rate
limiting (Fig. 2), and this effect was inhibited by the
Cl
channel blockers NPPB
and niflumic acid; and 3) the
currents activated by 2-chloroadenosine displayed a
Cl
-dependent
Erev and were
reversibly inhibited by the
Cl
channel blocker NPPB
(Figs. 5-8). Cl
channel activity of NPE cells has been posited to limit the rate of
secretion (8), so that the stimulation of
Cl
channels by adenosine
should accelerate the rate of aqueous humor formation.
How P1 purinergic receptors are
linked to Cl channels is
unclear. Occupancy of adenosine receptors
A1,
A2a,
A2b, and
A3 is thought to act at least
partly through decreasing or increasing the intracellular cAMP
concentration (15). However, the effects of very large concentrations
of adenosine (10 mM) and DBcAMP (0.5 mM) appeared to be additive (Fig.
4B), presumably reflecting
independent actions. Changes in PKC activity have also been thought to
mediate some of the effects of adenosine (22). However, blocking PKC activity with even a large concentration of staurosporine (0.3 µM)
was insufficient to replicate the adenosine-triggered stimulation (P < 0.001, Fig.
4A), whereas addition of the two
agents was no more effective than the adenosine alone
(P > 0.4). We conclude that some of
the actions of adenosine may be mediated through an inhibition of
endogenous PKC activity, but some must be mediated through a different
signaling pathway and/or by targeting a different population of
Cl
channels. Alternative
possible mediators include intracellular Ca2+ (14) and G proteins (15). The
latter possibility is particularly intriguing since all the known
mammalian adenosine receptors (A1, A2a,
A2b, and
A3) are coupled to G proteins
(15), and G proteins have been implicated in modulating
Cl
channel activity of
pigmented ciliary epithelial cells (18). These possibilities are
currently under study.
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ACKNOWLEDGEMENTS |
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We thank Dr. Martin Pring for helpful discussions concerning statistical analysis of the data.
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FOOTNOTES |
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This work was supported in part by National Eye Institute Grants EY-10691 and EY-00785 for core facilities. C. H. Mitchell was partially supported by Visual Sciences Training Grant 5-T32-EY-07035-17 and Respiratory Training Grant 2-T32-HL-07027-21 in successive years.
Some of these results have been presented in preliminary form (3, 4).
Address for reprint requests: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, Richards Bldg., Philadelphia, PA 19104-6085.
Received 3 September 1996; accepted in final form 25 June 1997.
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