Download Characterization of beta-adrenergic receptors in cultured

Investigative Ophthalmology & Visual Science, Vol. 30, No. 1, January 1989
Copyright © Association Tor Research in Vision and Ophthalmology
4*
Characterization of /?-Adrenergic Receptors in
Cultured Human Trabecular Cells and in
Human Trabecular Meshwork
Martin D. Wox,*f Perry B. Molinoff,f Jorge Alvarodo,^ ond Jon Polonsky^
The characterization of /S-adrenergic receptors on cultured human trabecular cells and trabecular
meshwork from human autopsy eyes was carried out by radioligand binding utilizing (l25!)-iodopindolol. In cultured cells, the observed binding of (I2SI)-iodopindolol was of high affinity (Kj = 43 pM) and
saturable. Scatchard plots were linear and revealed a Bmax of 33 ± 7 fmol/mg of protein. Competition
studies with a series of agonists and antagonists revealed that human trabecular cells contain a single
class of 0-adrenergic receptors of the 02 subtype. Similarly, the IQo of ICI 89,406 (176 nM) in human
trabecular meshwork from autopsy eyes supports the presence of /32-adrenergic receptors in this
tissue. Invest Ophthalmol Vis Sci 30:51-57,1989
The trabecular meshwork is the primary structure
through which aqueous humor must flow during its
egress from the anterior chamber of the eye. It is
composed of branching, interlacing collagenous
beams, each enclosed by a layer of endothelial cells.
The trabecular meshwork is one of several structures
in the anterior chamber angle through which filtration may occur. These include the juxtacanalicular
tissue, the canal of Schlemm, and the efferent venous
collector channels.1 Impaired drainage of aqueous
humor through these structures is thought to underlie
the primary pathophysiologic abnormality in openangle glaucoma, namely, diminished outflow facility
of aqueous humor from the anterior chamber.2-3
Only limited success has been achieved in finding
histologic or morphologic changes in the trabecular
meshwork or its adjacent structures that would account for the alterations in outflow of aqueous
humor that accompany glaucoma. The possibility
that changes in the mucopolysaccharide composition
of the trabecular meshwork, or in the mechanism by
which endothelial pinocytotic vesicles apparently
transport materials (including aqueous humor) into
the canal of Schlemm, has been studied in an effort to
explain the reduced outflow of aqueous humor,
hence elevated intraocular pressure, in glaucoma.
The trabecular meshwork/filtration apparatus is
also thought to be the site at which pharmacologic
agents act to improve the outflow facility of the eye.
Epinephrine, used for the treatment of glaucoma
since 1894,4 is thought to improve aqueous outflow
although the precise mechanism that underlies this
effect is not known.5"7 Specifically, it remains unclear
whether this effect is mediated primarily by a-adrenergic5'8"12 or 0-adrenergic receptors. 1314 These discrepancies may be partially due to species variability.
It has been suggested that in rabbits, both a- and
(8-adrenergic agonists increase outflow facility,
whereas in humans and monkeys there is no apparent
effect from stimulation of a-adrenergic receptors although an increase does occur after stimulation of
/3-adrenergic receptors. 6714
Further understanding of the pharmacologic properties and characteristics of the trabecular meshwork
has come from results of the in vitro study of human
trabecular cells.15"17 Trabecular cells obtained from
humans and grown in primary culture have been
used to study changes in intracellular cAMP production in response to catecholamines.15 Furthermore,
possible morphologic alterations have been observed
in these cells in response to prolonged administration
of epinephrine.18 These functional effects are thought
to be initiated following occupancy of j3-adrenergic
receptors by epinephrine on the trabecular cells.
We have characterized the pharmacologic properties of the /3-adrenergic receptors of human trabecular
cells grown in culture, and have compared their prop-
From the Departments of fPharmacology and 'Ophthalmology,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania, and the ^Department of Ophthalmology, University
of California, San Francisco, California.
Supported by USPHS Grants NS 18479, EY-06810, MH14654,
EY-03980, EY-02068, and EY-02162.
Submitted for publication: May 7, 1987; accepted April 19,
1988.
Reprint requests: Martin Wax, MD, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104-6084.
51
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1989
Vol. 30
v
Fig. 1. Photomicrograph of the trabecular meshwork region of a human eyefixed24 hr postmortem shows the region from which trabecular
tissue was obtained. After iris was removed from its insertion, gentle scraping was performed to obtain the tissue from Schwalabe's line to the
scleral spur (between the black arrows), and medial to the inner wall of the canal of Schlemm (SC) (X50).
erties to those observed in studies of trabecular meshwork tissue that was obtained from human eyes
shortly after death. Our findings indicate that the /Sadrenergic receptors of human trabecular cells in
vitro and in tissue from the trabecular meshwork in
vivo are characterized by the presence of/3-adrenergic
receptors of the /82 subtype.
Materials and Methods
Tissue Preparation
Freshly enucleated eyes from human donors (ages
56 to 75) were obtained within 24 hr of death due to
cardiorespiratory failure and were dissected on ice in
tissue buffer (20 mM Hepes buffer, pH 7.5, containing 145 mM NaCl). The pars plana was incised 4 mm
posterior to the corneascleral limbus, and the anterior
segment was removed intact by surgical separation.
After removal of the iris/ciliary body, the corneoscleral remnant, which included the trabecular meshwork, was scraped from the scleral spur to Schwalabe's line and placed in tissue buffer for further processing. Sections of the corneoscleral/trabecular
meshwork remnant were also sent for histopathologi-
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cal analysis to confirm the presence of trabecular
meshwork (Fig. 1). Tissues from human eyes were
pooled in tissue buffer, homogenized with a Brinkmann (Westbury, NY) Polytron (five times for 1 sec
at setting 6-7), and centrifuged at 20,000 g. Membranes were resuspended in tissue buffer and stored
frozen at —70°C until they were thawed for binding
assays. Tissues from seven eyes were thawed and
pooled for an additional wash at 20,000 g and resuspended in a suspension of 25% Percoll (Pharmacia
Fine Chemicals, Uppsala, Sweden) in 20 mM Hepes
buffer, pH 7.5, containing 145 mM NaCl. The Percoll suspension containing tissue was placed in a
Beckman Polyallomer (Palo Alto, CA) centrifuge
tube (16X76 mm) and centrifuged at 20,000 rpm in
a 70.1 Ti rotor to form a self-generated isopychnic
gradient. Centrifugation resulted in the separation of
plasma membranes from the melanin pigment-containing pellet at the bottom of the tube.19 The properties of/3-adrenergic receptors were then characterized
in binding assays that used the radioligand (l25I)-iodopindolol (125I-IPIN). (I25I)-IPIN was prepared by a
modification of the method of Barovsky and
No. 1
0-ADRENERGIC RECEPTORS IN TRADECULAR CELLS / W a x er al
53
Fig. 2. Human trabecular
cells grown postconfluency
as described in Methods
(X100).
Brooker,20 as described by Wolfe and Harden,21 and
used in binding assays to assess the properties of &adrenergic receptors in human eye tissue as previously described.19
Cell Culture
Human trabecular meshwork cells were obtained
from the eyes of a 30-year-old patient with no known
ocular disease and grown for 2 to 4 weeks postconfluency. This was approximately 1 week longer than
required to obtain optimally stable, morphologically
differentiated monolayers.15"17 Fourth-passage cells
from frozen stock were plated at 5000 cells/cm2 in
100 X 15 mm dishes (Falcon Labware, Oxnard, CA),
and grown in Dulbecco's modified Eagle's medium
(Gibco Laboratories, Grand Island, NY) containing 2
mM glutamine, 15 ng/rs\\ gentamycin, and 2.5 mg/ml
Fungizone. Medium was changed every 2-3 days and
was supplemented with 15% fetal calf serum (Sterile
Systems Inc., Logan, UT) during the growth phase to
confluency in an atmosphere of 10% CO2-90% air at
37 °C. In addition, 250 ng/ml of fibroblast growth
factor (FGF) was added every other day until confluency was achieved. At confluency, cells were subsequently maintained in 10% fetal calf serum and
grown in the absence of FGF for 10-14 days prior to
harvest (Fig. 2). The medium was changed 18-24 hr
prior to harvesting at which time cells were washed
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twice with phosphate-buffered saline and once with
20 mM Hepes buffer, pH 7.4, containing 145 mM
NaCl. Cells were removed with a rubber policeman
and centrifuged at 1500 g for 10 min. The supernatant was discarded and the pellet was stored at
—70°C. Before use, cells were thawed and resuspended in 20 mM Hepes buffer, pH 7.4, containing
145 mM NaCl. Membranes were prepared by homogenizing the cells with a Brinkmann Polytron at
setting 5-6 for 5 sec and centrifuging at 20,000 g for
15 min. The supernatant was discarded, and the
membranes were resuspended in buffer as above
prior to their addition to the binding assays.
Assay of /9-Adrenergic Receptors
Properties of /?-adrenergic receptors were assessed
by adding aliquots of crude membranes to assay tubes
containing, in a final volume of 0.25 ml, 12 mM
Hepes buffer and 0.5% NaCl, bovine serum albumin
(0.0004%) and ascorbic acid (1.1 mM). Assays were
carried out in triplicate in the presence of 100 //M
GTP. Specific binding was denned as the amount of
(125I)-IPIN bound in the absence of a competing ligand minus the amount bound in the presence of 100
nM (-)-isoproterenol. Equilibrium binding was performed for 20 min at 37°C in disposable polypropylene tubes (Walter Sarstedt, Princeton, NJ). Reactions
were terminated by dilution with 10 ml of buffer (10
54
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1989
mM Tris HC1, pH 7.5, containing 154 mM NaCl) at
room temperature. Samples were then filtered over
Schleicher and Schuell (Keene, NH) glass-fiber filters
(No. 30) on a millipore vacuum filter manifold. The
filters were washed with an additional 10 ml of buffer
at room temperature and dried with suction. Radioactivity was determined in an LKB 1274 or a Beckman 4000 gamma counter. Protein was determined
by the method of Bradford22 using bovine serum albumin as a standard.
Drugs
The following drugs were kindly provided as gifts:
ICI 118,551 and 89,406 (Imperial Chemical Industries America, Wilmington, DE). The 1-stereoisomers of isoproterenol, epinephrine, and norepinephrine were purchased from Sigma Chemical Co. (St.
Louis, MO).
Data Analysis
Saturation data were analyzed by the method of
Scatchard23 to provide an estimate of the density of
receptors and the affinity of the receptor (KJ for
(I25I)-IPIN. Dose-response curves for the inhibition
of the binding of (125I)-IPIN were analyzed by the
computer-aided curve-fitting program NEWFITSITES on the NIH-sponsored PROPHET computer
system. Using nonlinear regression analysis, the
mathematical modeling program MLAB was used to
identify the presence of one or two classes of binding
sites by comparing the sum of squares of the residuals
for one- and two-site models. A two-site model was
considered to fit the data significantly better than a
one-site model if an .Ftest of the sum of squares of the
residuals yielded a significance level of improvement
of fit of P < 0.001. Kd values for the binding of a
competing drug were calculated from the IC5o values
using the method of Cheng and Prusoff.24
Results
Direct Binding of Cultured Human Trabecular Cells
Direct binding of (125I)-IPIN to membranes from
cultured human trabecular cells was carried out with
six to eight concentrations of (125I)-IPIN ranging from
10 to 400 pM. The observed binding of (125I)-IPIN
was of high affinity (K<i = 43 ± 4 pM) and saturable
(Fig. 3). Scatchard plots were linear and revealed a
Bmax of 33 ± 7 fmol/mg of protein.
Analysis of Indirect Binding
The ability of several agonists and antagonists to
compete for binding sites for (I25I)-IPIN was deter-
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Vol. 30
mined. The affinities of drugs were assessed by incubating homogenates of cultured human trabecular
cells with 150 pM (l25I)-IPIN in the presence of 16 to
20 concentrations of competing drug. The relative
proportions of 0r and ft-adrenergic receptors were
determined by computer-assisted analysis of dose-response curves resulting from the inhibition of the
binding of (125I)-IPIN with 20 concentrations of the
j3rselective antagonist ICI 89,406 or the fo-selective
antagonist ICI 118,551. In the cultured trabecular
meshwork cells, the inhibition curves were best explained by the presence of a single population of
binding sites. ICI 118,551 was approximately 90-fold
more potent in displacing (125I)-IPIN from the (32adrenergic receptors in human trabecular cells than
was ICI 89,406 (Fig. 4). The observed order of potency for the inhibition of the binding of (I25I)-IPIN
in vitro by agonists was isoproterenol > epinephrine
> norepinephrine (Fig. 5, Table 1).
A similar assay for (I25I)-IPIN binding sites was
performed in postmortem human trabecular tissue
pooled from seven patients. The results obtained
were similar to those found in vitro since the inhibition curve was best fit by a model that assumes the
presence of one class of binding sites. The IC50 value
of ICI 89,406 in displacing specifically bound (125I)IPIN from postmortem human trabecular tissue was
176 nM and is similar to that obtained in the human
trabecular cells grown in tissue culture (Fig. 6). Using
the assumption that the affinity of the radioligand
(I25I)-IPIN is similar in both human trabecular tissue
and cultured cells, we may estimate that the density
of receptors in the human trabecular meshwork is
approximately 70 fmol/mg.
Discussion
The ability to propagate human trabecular cells in
serial culture facilitates the study of receptor-mediated hormone interactions at the level of the cell
membrane that cause subsequent biochemical responses in this tissue. The presence of saturable specific binding of (I25I)-IPIN demonstrates the conservation of membrane-bound /3-adrenergic receptors in
broken cell preparations of human trabecular cells
grown in tissue culture.
|8-Adrenergic receptors have been classified into
two subtypes based on the pharmacologic specificity
of adrenergic receptor-mediated responses to selective drugs.25 Analysis of the subtypes of membranebound /3-adrenergic receptors can be carried out by
using radioligand binding assays in which selective
antagonists compete for specifically bound (I25I)IPIN.26 In our experiments, 0-adrenergic receptors on
trabecular cells grown in culture had a high affinity
No. 1
55
/3-ADRENERGIC RECEPTORS IN TRADECULAR CELLS / W a x er al
Fig. 3. Density of /3adrenergic receptors in
human trabecular cells. A
representative saturation
curve for binding of membranes of human trabecular
cells grown in culture to
(125I)-IPIN is shown. Total
binding (•), specific binding (A), and nonspecific
binding (•) are shown
in addition to the linear
Scatchard transformation
(inset).
TOTAL
LU
o
d
Q_
5X 10 - 1 0
- IPIN (M)
for the ft-selective antagonist ICI 118,551, and a low
affinity for the /?,-selective antagonist ICI 89,406.
This is consistent with the reported properties of the
/32 subtype of the /3-adrenergic receptor. The potency
of ICI 89,406 at /3-adrenergic receptors in human trabecular tissue from postmortem eyes was similar to
that which we observed in vitro. Furthermore, in
both tissues, only a single homogeneous class of binding sites was found.
Although considerably more efforts are necessary
to define the mechanism(s) by which adrenergic
drugs facilitate outflow, our findings provide some
80
80
60
o
potentially useful information. Physiological studies
in monkeys suggest that outflow facilitated by adrenergic agonists is mediated by /3-adrenergic receptors
since cyclic AMP was elevated in aqueous humor
with agonist stimulation and the response was
blocked by propranolol.67 Similarly, clinical investigation in humans suggested that timolol was able to
block the ability of epinephrine to increase outflow
facility.13 The use of the nonselective /3-adrenergic
antagonists propranolol and timolol, however, does
not provide useful information concerning the /3adrenergic receptor subtype that mediates their ef-
60
ICI 1 18.551O
40
20
20
o
11.
9
7
-LOG DRUG (M)
5
Fig. 4. Inhibition of the binding of (I25I)-IPIN by selective antagonists in membranes of cultured human trabecular cells. Assays
were carried out with 100 pM (I251)-IPIN. The IC50 value and Hill
coefficient were 3.5 nM and 0.94 for ICI ! 18,551, and 270 nM and
0.90 for ICI 89,406. The data are from a representative experiment
performed in triplicate.
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-LOG DRUG (M)
Fig. 5. Inhibition of the binding of (I25I)-IPIN by selective agonists in membranes of cultured human trabecular cells. Assays were
carried out with approximately 140 pM (125I)-IPIN. The IC50 values
for isoproterenol (0.9 nM), epinephrine (4.7 /iM), and norepinephrine (18.7 tiM) are the means of triplicate determinations. The Hill
coefficients for the agonists were each approximately 0.9. The data
are from a representative experiment performed in triplicate.
56
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Jonuory 1989
Table 1. Apparent equilibrium constants for drugs
acting at /3-adrenergic receptors in human
trabecular cells
Drug
Antagonists
ICI 118,551
ICI 89,406
Agonists
Isoproterenol
Epinephrine
Norepinephrine
Selectivity
ft
/3,
nonselective
nonselective
Kj ± SEM
(nM)
1.0 ±
91.0 ±
0.1
0.1
163 ± 20
1165 ± 65
4780 ± 270
Competition experiments for binding sites for (17!I)-IPIN were performed
as described in Materials and Methods. All assays were performed in the
presence of 100 nM GTP and represent the average of at least two experiments performed in triplicate. The order of potency is characteristic of the fe
subtype of the /3-adrenergic receptor.
fects. Recent reports have suggested that the /Si-selective antagonist betaxolol may not fully block epinephrine's effect on outflow facility in humans. 27
This observation may be partially explained by our
finding that the /3-adrenergic receptors in trabecular
cells and trabecular meshwork are of the /32 subtype,
and is consistent with the finding that betaxolol (10~6
M) does not fully block epinephrine stimulation of
cyclic AMP production in cultured trabecular cells.28
The relative affinities of drugs selective for /3adrenergic receptors in these tissues may have an important bearing on the efficacy and safety of topically
administered /3-adrenergic antagonists used in the
treatment of glaucoma. The finding that the /3-adrenergic receptors in human trabeculum are of the 182
subtype is consistent with results of previous investigations.19 It is reasonable to assume that side effects
due to systemic absorption of topically applied /?adrenergic receptor antagonists may be minimized by
using the lowest concentration of drug that effectively
-LOG ICI 89,406 (M)
Fig. 6. Inhibition of the binding of (125I>IPIN to /3-adrenergic
receptors by ICI 89,406 in human trabecular tissue obtained from
postmortem eyes. Assays were carried out with approximately 70
pM of (12SI)-IPIN. The observed IC50 value of 176 nM represents
the results of triplicate determinations from tissue pooled from
seven eyes. The Hill coefficient was 1.07, and the data were best fit
to a model suggesting the presence of a single class of binding sites.
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Vol. 30
occupies the ocular receptors responsible for mediating the hypotensive effect and results in effective lowering of intraocular pressure. Certainly there are multiple factors in addition to potency, such as systemic
absorption, protein binding, volume of distribution,
and rate of elimination which may influence ocular
bioavailability as well as the observed systemic side
effects of these drugs.
Apparently /3-adrenergic antagonists such as timolol effectively reduce intraocular pressure by decreasing aqueous inflow. This response is presumably the
result of antagonist occupancy of /32-adrenergic receptors located on the ciliary epithelium of the ciliary
processes. The use of drugs, therefore, which have a
high potency for /32-adrenergic receptors in the eye
may allow effective reduction of intraocular pressure
while minimizing cardiovascular side effects such as
heart block and congestive failure which are thought
to be due primarily to /3,-adrenergic receptor blockade. On the other hand, when we speculate as to the
role of j8-adrenergic receptor occupancy on outflow
facility we must consider the possibility it may not be
therapeutically desirable to achieve /3-adrenergic
blockade in the trabecular meshwork which could
result in decreased outflow facility when epinephrine
is used concomitantly. Towards that objective, it may
be advantageous to use a jSi -selective antagonist at a
concentration which results in incomplete occupancy
of ft-adrenergic receptor sites in the outflow pathways. Unfortunately, it is difficult to explain how a
/?i -adrenergic antagonist, at a concentration which
fails to occupy /32-adrenergic receptors in the outflow
pathway, could thus effectively occupy the /32-adrenergic receptors on the ciliary epithelium to effect inflow. It is certain that further work is necessary to
fully explain the cellular mechanisms by which occupancy of specific receptors effect the regulation of
aqueous secretion by the ciliary epithelium and outflow via the trabecular meshwork. Such considerations, however, are certainly important in understanding the rationale for the current and future use
of/3-adrenergic receptor agonists or antagonists in the
treatment of glaucoma.
In conclusion, we have demonstrated that trabecular cells grown in culture possess /3-adrenergic receptors of the /32 subtype which are similar to those found
in trabecular tissue in vivo. As such, the further study
of trabecular cells grown in culture appear to be
worthy of pursuit in the hope of providing insight
into the mechanism by which several families of
drugs modulate the function of trabecular cells, thus
clarifying the rationale for their use in glaucoma.
Key words: trabecular cells, trabecular meshwork, j8-adrenergic receptors, l25I-iodopindolol
No. 1
/8-ADRENERG1C RECEPTORS IN TRABECULAR CELLS / W a x er al
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