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Investigative Ophthalmology & Visual Science, Vol. 33, No. 2, February 1992
Copyright © Association for Research in Vision and Ophthalmology
The Influence of Cyclic AMP Upon Na,K-ATPase
Activity in Rabbit Ciliary Epithelium
N. A. Delamere and K. L. King
ATPase activity was measured in samples of freshly dissected rabbit ciliary epithelium. The epithelium
was ruptured in distilled water, frozen briefly, and incubated at 37°C in a buffer containing 100 mM
NaCl and 32P-labeled adenosine triphosphate (ATP). The rate of ATP hydrolysis by the epithelium was
linear for as long as 45 min. Ouabain (1 mM) reduced the ATP hydrolysis rate by approximately 50%.
When the epithelium was preincubated for 10 min. in the presence of 1 mM dibutyryl cyclic adenosine
monophosphate (cAMP), the ouabain-sensitive (Na,K-ATPase) activity was diminished; ouabain-insensitive ATPase activity was not reduced. Preincubation of the epithelium with forskolin with isobutylmethylxanthine also reduced ouabain-sensitive ATPase activity. These observations suggest that
the ciliary epithelium may have a mechanism for short-term modulation of Na,K-ATPase activity by
cAMP. Such a mechanism could be linked to the ability of cAMP-dependent protein kinase to reduce
Na,K-ATPase activity in the tissue. Invest Ophthalmol Vis Sci 33:430-435,1992
Mittag et al6 have obtained direct evidence that the
rabbit ciliary epithelium has adenylate cyclase that is
vasoactive intestinal peptide and beta-adrenergically
responsive. These findings suggest that cAMP might
have a role in the transduction of signals within the
epithelium. In a recent study, we isolated membrane
material from rabbit ciliary processes and observed
that Na,K-ATPase activity in the preparation was diminished in the presence of cAMP-dependent protein
kinase, protein kinase A.7 This finding suggested that
when cytoplasmic cAMP activates protein kinase A,
Na,K-ATPase activity could be lowered, perhaps signifying that cAMP is involved in short-term regulation of Na,K-ATPase activity in rabbit ciliary processes. A role for cAMP in short-term regulation of
Na,K-ATPase activity in kidney proximal tubule segments has been proposed by Bertorello and Aperia.8
In the present study, we have examined changes of
Na,K-ATPase activity that occur when the ciliary epithelium is pretreated with dibutryl cAMP or forskolin. To do this, we isolated pure samples of rabbit
ciliary epithelium and established a protocol for measuring Na,K-ATPase activity in permeabilized cells.
This approach differs from the conventional methodology of determining Na,K-ATPase activity in a purified membrane preparation made by a lengthy differential centrifugation procedure, during which loosely
bound membrane-associated components may be
lost from the epithelial membranes. Using the permeabilized cell technique, ATPase activity could be
measured immediately after the epithelium was removed from the incubation solution.
The ciliary processes are covered by a doublelayered epithelium, the ciliary epithelium, which is
recognized as the site of aqueous humor formation.1
The ciliary epithelium has a high Na,K-ATPase activity, and morphological studies have shown that the
Na,K-ATPase is most densely localized at the basolateral membranes of the nonpigmented epithelium, the
epithelial layer that faces the aqueous humor.2 While
the precise contribution of active sodium transport to
aqueous secretion has yet to be elucidated, it is widely
believed that active sodium transport is linked to the
mechanism of fluid formation by the epithelium.1 In
fact, experiments by Cole,3 Becker,4 and Bonting and
Becker5 have shown that aqueous humor formation
in rabbits can be inhibited by ouabain, the specific
inhibitor of Na,K-ATPase.
It has been known for some time that intraocular
pressure can be lowered by a number of agents that
alter the level of cyclic adenosine monophosphate
(cAMP) in tissues at the anterior of the eye (Davson1).
From the Department of Ophthalmology and Visual Sciences,
Kentucky Lions Eye Research Institute, University of Louisville
School of Medicine, Louisville, Kentucky.
Supported by USPHS Research Grant No. EY06915, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc., New York, New York.
Submitted for publication: March 12, 1991; accepted September
19, 1991.
Reprint requests: Nicholas A. Delamere, Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville,
KY 40292.
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NA,K-ATPA5E ACTIVITY IN CILIARY EPITHELIUM / Delomere ond King
Materials and Methods
Chemicals
Ouabain, dibutyryl cAMP (N6,2'-O-dibutyryl adenosine 3', 5'-cyclic monophosphate), forskolin, and
IBMX (isobutylmethylxanthine) were obtained from
Sigma (St. Louis, MO). ATP as a triethylammonium
salt labeled with 32P at the terminal phosphate group
was purchased from Amersham (Arlington Heights,
IL). All other chemicals were purchased from Fisher
(Pittsburgh, PA) or Sigma.
Isolation of Ciliary Epithelium
Ciliary epithelium was obtained from adult New
Zealand White rabbits weighing 2-3 kg that were sacrificed by an overdose of sodium pentobarbital administered through a marginal ear vein. The use of animals in this study conformed to the ARVO Resolution on the Care and Use of Animals in Research.
After death, the eye was proptosed and the cornea was
removed. The iris, ciliary body, lens, and vitreous
body then were removed from the eye in one piece
431
and transferred to a dish of ice-cold Kreb's solution
containing 119 raM NaCl, 4.7 mM KC1, 1.1 mM
KH2PO4> 1 mM MgCl2; 5.5 mM glucose, and 25 mM
NaHCO3 at pH 7.4. The Kreb's solution was pregassed with 95% 0^5% CO2. The ciliary epithelium
was dissected from the eye using a method described
in detail by Jumblatt and coworkers.9 A radial cut was
made through the iris from the pupil to the periphery.
The iris and base of the ciliary body then was gently
peeled away from the lens, leaving the ciliary epithelium from the ridges and valleys of the pars plicata
attached to the lens zonules (suspensory ligaments).
Finally, the lens zonules were cut, freeing the ciliary
epithelium as a single strip of tissue 25-35 mm long.
The ciliary epithelium from each eye was divided into
five pieces.
ATPase Assay
The ATPase assay was developed to measure ATP
hydrolysis by the ciliary epithelium shortly after an
incubation period in Kreb's solution. The assay
method is a modification of a technique used by Katz
Fig. 1. The rabbit ciliary epithelium immersed in Krebs solution.
Viewed at a magnification of X100 (A), ridges can be seen at the
points where the epithelium had covered the ciliary processes. At
higher magnification (X400) using Nomarski optics, the nonpigmented cells are shown in freshly dissected tissue (B). A low-power
transmission electron micrograph of the tissue (C) shows the two
cell layers.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / February 1992
and coworkers.10 It differs from conventional ATPase
assays because ATPase activity is measured in permeabilized cells rather than in membrane material
that is first isolated from the tissue and purified by
several stages of washing and centrifugation.
After a specified period of incubation in Kreb's solution, the ciliary epithelium was removed and permeabilized in a two-stage process. First, each piece of
epithelium was placed in a tube containing 184 n\
distilled water for 5 min so the cells could swell and
rupture. Permeabilization was ensured by a brief
freeze-thaw in liquid nitrogen before 166 /x\ of a buffer
solution was added to give a final composition of 40
mM histidine HC1, 10 mM KC1, 3 mM mg MgCl2,
100 mM NaCl and 0.8 mM Tris-EGTA and 1 mM
32
P-labelled ATP at pH 7.4. The buffer solution also
contained 11 /ig/ml alamethicin, which was added to
ensure a high cation permeability of any resealed
membrane compartments and to counter the possibility that Na,K-ATPase activity would be influenced by
local cation gradients.11 In specified experiments, the
solutions contained 1 mM ouabain. This buffer composition is the same as that used earlier for experiments with partially purified ciliary process membrane preparations.7
The assay mixture was incubated at 37°C for a specified period, and the reaction was terminated by the
addition of 100 ^1 ice cold 0.5 NaOH using an established methodology.12 ATP hydrolysis in each tube
was quantified by determining the amount of radiolabelled inorganic phosphate that had been released.
The amount of tissue protein in each tube was determined by a Bio Rad assay. ATPase activities were calculated as nanomoles phosphate released//tg protein/
h. A paired Student's t test was used to determine the
statistical significance of differences between ATPase
activities measured in treated and control tissues.
Vol. 33
layer. The epithelial layers remain attached to the lens
zonules when the iris and ciliary body are stripped
away. The epithelium is removed as a narrow sheet
that is wrinkled at the points where it used to fold over
the surface of the ciliary processes (Fig. 1).
ATPase Activity
ATPase activity was measured in freshly dissected
ciliary epithelium following a brief permeabilization
procedure. The rate of ATP hydrolysis was linear over
a period of 45 min. (Fig. 2). The control rate of ATP
hydrolysis was 4.1 ±0.6 nmol phosphate released//ig
protein/h (mean ± SEM). In the presence of 1 mM
ouabain, the rate was 2.0 ± 0.2 nmol phosphate released//^ protein/h, which is significantly different (P
= .001) from the control value.
Influence of Dibutyryl cAMP Upon ATPase Activity
Dibutyryl cAMP was used because it is more permeable than cAMP. Freshly dissected ciliary epithelium
was maintained for 10 min at 37°C in Kreb's solution
containing 1 mM dbcAMP. Control pieces of ciliary
epithelium were incubated in the absence of
dbcAMP. After the incubation period, the pieces of
epithelium were removed from the Kreb's solution
immediately prior to permeabilization and measurement of ATP hydrolysis. Thus, dbcAMP was not present during the ATP hydrolysis portion of the experiment.
In epithelium pretreated with dbcAMP, the ouabain-sensitive portion of ATP hydrolysis was reduced.
CONTROL
Results
Isolation of Ciliary Epithelium
In the past, most ATPase measurements and related transport studies were conducted with segments
of iris-ciliary body or with ciliary processes as a source
of tissue.712 These tissues contain a number of different cell types. In the present study, we have used a
technique in which pure ciliary epithelium is isolated
from the ridges and valleys of the pars plicata. The
dissection procedure, described in detail by Jumblatt
and coworkers9, relies on the close attachment of the
lens zonules with the basement membrane of the nonpigmented (aqueous humor facing) ciliary epithelium
OUABAIN
60
TIME (min)
Fig. 2. Hydrolysis of ATP by permeabilized ciliary epithelium in
the presence (closed circles) or absence (open circles) of ouabain.
Each point represents the mean of data from 12 tissue pieces with
the standard error indicated by a vertical line. The lines were fitted
to the data by linear regression.
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NA,K-ATPASE ACTIVITY IN CILIARY EPITHELIUM / Delomere and King
No. 2
A typical experiment is shown in Figure 3a. In eight
experiments, pretreatment of the epithelium with 1
mM dbcAMP reduced the total ATP hydrolysis rate
by slightly more than 50% (P < 0.01), while the ouabain-insensitive ATPase component was not significantly altered (Fig. 3b). No significant alteration of
ATPase activity was observed when the epithelium
was pretreated with 10~4 M or 10"5 M dbcAMP.
We also examined the influence of forskolin on
ATPase activity because concentrations of 10"5 and
10~4 M forskolin activate adenylate cyclase in rabbit
ciliary processes.13 ATPase activity was measured following a 10 min period of exposure of the ciliary epithelium to forskolin (5 X 10~5 M) in Kreb's solution
that also contained the phosphodiesterase inhibitor
IBMX (10"4 M). Forskolin/IBMX caused a reduction
of total ATPase activity, but ouabain-insensitive ATPase activity was not affected (Fig. 4).
Control epithelium
dbcAMP-treated epithelium
433
C Z l - OUABAIN
^
+ OUABAIN
120
100
( .b
80
< 8
60
O
c
r-i
40
20
•
CONTROL
10 min PRETREATMENT
FORSKOLIN/IBMX
(5X 105M/10"\i)
Fig. 4. The influence of forskolin + isobutylmethylxanthine
(IBMX) upon ATPase activity measured in ciliary epithelium. Tissues were preincubated for 10 min in control Krebs solution or
Kreb's containing forskolin (5 X 10"5 M) together with IBMX (10""
M). ATPase activity was determined in the absence (open bars) or
presence (shaded bars) of ouabain. The data are the mean of eight
experiments with the standard error indicated by the vertical bar.
Total ATPase activity ( - ouabain) was significantly lower (P
< 0.01) in forskolin/IBMX-treated ciliary epithelium. Ouabain-insensitive ATPase activity (+ ouabain) was not significantly altered
by forskolin/IBMX pretreatment.
Discussion
0
10 20 30 40 SO 60 70
TIME (min)
0
10 20 30 40 SO 60 70
TIME (min)
C H - OUABAIN
+ OUABAIN
CONTROL
10 min PRETREATMENT
WITH 1 mM dbcAMP
Fig. 3. The influence of dibutryl cAMP upon ATP hydrolysis. (A)
A typical experiment showing the ATP hydrolysis by pieces of ciliary epithelium preincubated for 10 min in control Krebs solution
(left panel) or Krebs solution containing 1 mM dbcAMP (right
panel). Each point shows the ATP hydrolysis measured from a single tissue piece. (B) The mean rates of ATP hydrolysis measured in
control and dbcAMP-treated ciliary epithelium in the presence
(shaded bar) or absence (open bar) of ouabain. The data of the
mean of eight experiments with the standard error indicated by the
vertical bar.
The present experiments demonstrate that when
rabbit ciliary epithelium from the pars plicata is pretreated with dibutyryl cAMP, ouabain-sensitive ATPase activity is reduced. Ouabain-insensitive ATPase
activity is not diminished by dbcAMP pretreatment.
Forskolin, an activator of adenylate cyclase, elicits
similar results. While cAMP might alter many aspects
of cellular function in the ciliary epithelium, our finding that dbcAMP or forskolin pretreatment reduces
Na,K-ATPase activity adds support to Mittag's proposal6 that the adenylate cyclase system in ciliary epithelial cells might participate in the regulation of the
membrane transport machinery involved in aqueous
humor secretion. Because cAMP also may cause parallel increases or decreases in the activity of other membrane transport mechanisms in the ciliary epithelium,
we are unable to specify what net changes of aqueous
humor formation would result following cAMP-dependent reduction of Na,K-ATPase activity. However, there have been reports of reduced aqueous humor flow in forskolin-treated rabbits and humans.13
The design of these studies resulted in ATPase activity being measured shortly after removal of the epithelium from Kreb's solution that contained dbcAMP or
forskolin. Thus, the modification of the ATP hydrolysis pattern, which was triggered by dbcAMP or forskolin, did not require the continued presence of these
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1992
agents. One possible explanation of these finding is
that cAMP alters epithelial membrane permeability,
and this change somehow alters the access of sodium
or potassium to the Na,K-ATPase. However, this interpretation seems unlikely because the ATP hydrolysis measurements were carried out in the presence of
alamethicin, which establishes high membrane cation
permeability.11 Therefore, it seems possible that
the diminished ouabain-sensitive ATPase activity
in dbcAMP-treated tissue may signify a cAMPdependent mechanism for short-term reduction of
Na,K-ATPase activity in the rabbit ciliary epithelium.
The existence of a mechanism for short-term regulation of Na,K-ATPase activity in rat kidney proximal
convoluted tubules (PCT) has been proposed by Bertorello and Aperia.8 Inhibition of PCT Na,K-ATPase
by dopamine14 and the demonstration that GTP binding proteins and protein kinase C may be involved1516
have led Bertorello and Aperia to propose a complex
model for the Na,K-ATPase regulatory system8. Because PCT Na,K-ATPase activity is reduced by specific DA 1 (dopaminergic) agonists17 and the effect can
be potentiated by dbcAMP or forskolin,18 activation
of adenylate cyclase and an increase in cellular cAMP
were suggested to be an integral part of the
Na,K-ATPase regulatory process. A similar pattern of
altered Na,K-ATPase activity was found recently in
guinea pig striatal neurons that had been exposed to
dopamine, specific D, and D2 dopamine receptor agonists, and forskolin.19 Interestingly, cAMP has been
reported to stimulate the active transport of sodium
and potassium in frog retinal pigment epithelium2021
and dogfish rectal gland.22
In many cases, cellular responses to cAMP are mediated by cAMP-dependent protein kinase23. This kinase, but not cAMP, reduces the Na,K-ATPase activity measured in a membrane preparation purified
from rabbit ciliary processes.7 In the present experiments with intact rabbit ciliary epithelium, cAMP-dependent protein kinase activation may be involved in
the mechanism that translates dbcAMP or forskolin
exposure into a change of Na,K-ATPase activity.
Experiments by Lingham and Sen24 and by Meister
et al17 support the notion that cAMP-dependent protein kinase might be a part of a mechanism that is able
to modulate Na,K-ATPase activity. However, how
activation of cAMP-dependent protein kinase could
result in a change of Na,K-ATPase activity has yet to
be determined. While Yeh and coworkers25 have suggested that Na,K-ATPase can be phosphorylated by a
membrane-bound protein kinase, and Lowndes et
al26 have shown that protein kinase C can induce
phosphorylation of Na,K-ATPase, there is no convincing evidence that phosphorylation of the
Vol. 33
Na,K-ATPase subunits by cAMP-dependent protein
kinase takes place or that such phosphorylation
would alter the activity of the enzyme as an ion transporter. Phosphorylation of an intermediate protein
may be required to bring about a change in
Na,K-ATPase activity. It has been suggested recently
that DARPP-32, a cAMP-regulated phosphoprotein
found in kidney27 and ciliary epithelium.28 may
be involved, possibly as a third messenger, in
Na,K-ATPase modulation.19
Key words: ciliary epithelium, cAMP, Na,K-ATPase
References
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16. Bertorello A and Aperia A: Regulation of Na+,K+-ATPase activity in kidney proximal tubules: Involvement of GTP binding
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Physiological Sciences, Helsinki, Finland, July 1989, p. 368.
19. Bertorello A, Hopfield J, Aperia A, and Greengard P: Inhibition by dopamine of (Na+ + K+)ATPase activity in neostriatal
neurons through D, and D2 dopamine receptor synergism. Nature 347:386, 1990.
20. Miller S and Farber D: Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. J Gen Physiol
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23. Berridge MJ: The molecular basis of communication within
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25. Yeh L, Ling L, English L, and Cantley L: Phosphorylation of
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28. Stone RA, Laties M, Hemmings HC, Ouimet CC, and Greengard P: DARPP-32 in the ciliary epithelium of the eye: A neurotransmitter-regulated phosphoprotein of brain localizes to secretory cells. J Histochem Cytochem 34:1465, 1986.
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