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2 Adrenergic Receptor Subtypes in Human Ocular Tissue Homogenates
From the Departments of 1 Pharmacology and 2 Ophthalmology, University of Nebraska Medical Center, Omaha.
Abstract
PURPOSE. To determine the predominant 
2 adrenergic receptor
subtypes present in the human eye.
METHODS. Saturation- and competition-receptor–binding experiments were
performed with the radioligand [3H]RX821002 in human
ciliary body, retinal pigmented epithelium–choriocapillaris, iris, and
neurosensory retina. The affinities of various adrenergic antagonists
in these ocular tissues were compared with their affinities for the
cloned 2A, 2B, and 2C
adrenergic receptor subtypes.
RESULTS. The density of 2 adrenergic receptors was highest in the
iris (440 femtomoles/mg protein), lowest in the neurosensory retina (14
femtomoles/mg protein), and intermediate in the other two tissues
(approximately 90 fmol/mg protein). The drug affinities in all four
human ocular tissues were highly correlated (correlation coefficients
between 0.94 and 0.97) with the affinities for the human
2A adrenergic receptor subtype and poorly correlated
(correlation coefficients between 0.15 and 0.66) with the
2B and 2C subtypes.
CONCLUSIONS. In agreement with previous studies in several animal species, the
2 adrenergic receptors in the human ciliary body,
retinal pigmented epithelium–choriocapillaris, iris, and neurosensory
retina are predominately of the 2A
subtype.
Glaucoma is characterized by a progressive loss of visual
sensitivity resulting from optic nerve damage. Because high intraocular
pressure is the most important risk factor for glaucoma, the treatment
of glaucoma has emphasized the reduction of intraocular
pressure.1
Alpha-2 adrenergic agonists such as brimonidine
and apraclonidine are effective ocular hypotensive
agents,2
3
4
although their mechanism of action is not
clear.5
6
7
The development of subtype-selective
2 adrenergic agents for topical application is desirable
to reduce both systemic and ocular side effects. An understanding of
the distribution of 2 receptor subtypes in the eye would
be useful in designing new drugs with greater effectiveness and fewer
adverse effects.
Based on both pharmacologic and molecular evidence, there are
three major types of adrenergic receptors, 1,
2, and ß, each of which is further divided into three
or four subtypes.8
The evidence for 2
adrenergic receptor subtypes has come from binding and functional
studies in various tissues and cell lines and more recently in cells
transfected with the cDNA for the receptors.9
On the basis
of these studies, three 2 adrenergic receptor subtypes
have been defined. The 2A adrenergic receptor subtype,
for which prazosin has a relatively low affinity and oxymetazoline a
relatively high affinity, is found in the human platelet and the HT29
cell.8
The second subtype, the 2B, was
identified in neonatal rat lung and in the NG108 cell.10
This subtype has a relatively high affinity for prazosin and a low
affinity for oxymetazoline. A third subtype, the 2C, has
been identified in an opossum kidney cell line.11
Although
this subtype also has a relatively high affinity for prazosin and a low
affinity for oxymetazoline, it is pharmacologically distinct from the
2B subtype.12
All three subtypes have been
cloned from the human.13
14
15
Using the homogenate
radioligand-binding technique, 2 adrenergic receptors
have been identified in ocular tissues of several species, including
the ciliary body, retinal pigmented epithelium (RPE)–choriocapillaris,
iris, and neurosensory retina of both the cow16
17
and the
pig18
and the ciliary body of the rabbit.19
The results of these binding studies indicate that the
2A subtype is the predominant, if not the only,
2 adrenergic subtype in these ocular tissues, with the
exception of the porcine neurosensory retina, which may contain a very
low density (4 femtomoles/mg protein) of the 2C subtype.
Although similar binding studies have not yet been conducted with human
ocular tissues, it appears likely that the predominant human ocular
2 adrenergic subtype may also be the 2A.
In contrast to these binding data, immunofluorescence labeling of the
human ciliary body indicates the presence of 2B and
2C subtypes, but not the 2A
subtype.20
Similarly, studies using polymerase chain
reaction (PCR) suggest the presence of the 2B and
2C subtypes, but not the 2A subtype, in a
transformed cell line of human nonpigmented
epithelium20
and only the 2B subtype
in the human ciliary body.21
To determine whether this
apparent discrepancy is the result of species differences or of
differences in techniques, we investigated the 2
subtypes in human ocular tissues by the radioligand-binding
technique. On the basis of receptor-binding experiments using the
2 antagonist radioligand
[3H]RX821002, we conclude that the
2 adrenergic receptors in the human ciliary body,
RPE–choriocapillaris, iris, and neurosensory retina are predominately
of the 2A subtype.
Methods
Drugs and Chemicals
[3H]RX821002 (specific activity 52–59
Ci/millimole) was obtained from Amersham International (London, UK);
rauwolscine, WB 4101, and spiroxatrine from Research Biochemicals,
(Natick, MA); and oxymetazoline from Sigma (St. Louis, MO). Prazosin
and ARC-239 were generous gifts from Pfizer (Groton, CT) and
Boehringer–Ingelheim (Ridgefield, CT), respectively. Drugs were
prepared as 5- or 10-mM stock solutions and
diluted in 5 mM HCl. The stock solution for prazosin was in methanol,
for spiroxatrine in 80% dimethyl sulfoxide-20% 1 M HCl, and for all
other drugs in 5 mM HCl.
Tissue
Human eyes were obtained frozen from the Missouri Lions Eye Bank
and bisected at approximately 7 to 8 mm posterior to the limbus while
bathed in 50 mM Tris buffer at 4°C. Under a dissecting microscope,
the anterior hyaloid face and lens material were carefully dissected
from the iris and ciliary body. The remnants of neurosensory retina,
RPE, and choriocapillaris were removed from their attachments at the
ora serrata in the anterior portion of the bisected globe. The iris was
then removed by disinserting the iris root from the base of the ciliary
body, thus releasing the base of the ciliary body then released from
its attachment to the scleral spur anteriorly.
The various tissues were suspended in 25 ml ice-cold 50 mM Tris-HCl (pH 8 at 25°C) and homogenized (model TR-10 Tissumiser; Tekmar, Cincinnati, OH). The homogenate was filtered through a 53-µm nylon mesh, centrifuged at 1400 rpm for 10 minutes. The supernatant was transferred to another tube, recentrifuged at 20,000 rpm for 10 minutes and the pellet frozen at -80°C.
Radioligand-Binding Assays
Saturation- and competition-binding experiments were performed as
described previously, using 25 mM sodium phosphate buffer at pH
7.4.16
17
22
Briefly, saturation experiments were
performed using two sets of duplicate tubes that contained 970 µl of
membrane suspension and 20 µl [3H]RX821002.
The protein concentration was adjusted to ensure that the specifically
bound radioligand was less than 10% of the total added radioligand.
One set of tubes contained 10 µl (-)-norepinephrine (final
concentration, 100 µM) to determine nonspecific binding. Specific
binding was calculated as the difference between total and nonspecific
binding. After a 40-minute incubation at room temperature, the
suspensions were filtered through glass fiber filter strips (GF/B;
Whatman, Clifton, NJ), which had been soaked overnight in 0.1%
polyethylenimine, using a 48-sample manifold (Brandel Cell Harvester;
Biomedical Research and Development, Gaithersburg, MD). The tubes and
filters were washed twice with 5 ml ice-cold 50 mM Tris-HCl (pH 8.0),
and the radioactivity on the filter was determined by liquid
scintillation spectroscopy. The Kd and
maximum binding (Bmax) values were
calculated from nonlinear regression of bound versus free ligand
concentrations using a statistical software program (Prism; GraphPad,
San Diego, CA). Kd values are
geometric means and
Bmax values are
arithmetic means. Protein concentrations were determined by the method
of Bradford23
with bovine serum albumin as the standard.
For inhibition experiments, 20 µl of a fixed concentration of radioligand [3H]RX821002 (final concentration, 0.24 ± 0.04 nM, which is near the Kd concentration) and various concentrations of unlabeled drug (10 µl) were added to duplicate tubes containing 970 µl of the membrane suspension. Assays were then performed as described for saturation experiments. Competition binding data were analyzed (Prism; GraphPad) to determine the 50% inhibitory concentration (IC50) assuming a one-site model. The pseudo Hill slope was determined by fitting the data to the four-parameter logistic equation. In some cases, the fit of the data to a one-site model was compared with the fit to a two-site model. IC50 values were converted to Ki values by the method of Cheng and Prusoff24 and are presented as geometric means.
Results
The selective 2 adrenergic antagonist
[3H]RX821002 demonstrated saturable and
high-affinity binding to membrane preparations from human ocular
tissues (Fig. 1
A). The density of receptor-binding sites
(Bmax) was highest in the iris,
intermediate in the ciliary body and RPE–choriocapillaris, and lowest
in the neurosensory retina (Table 1)
. The density was 30 times higher in the iris than in the
neurosensory retina, and 5 times higher than in the ciliary body and
RPE–choriocapillaris. The data were linear when plotted by the method
of Rosenthal,25
consistent with [3H]RX821002
binding to a single class of sites (Fig. 1B)
. The affinity
(Kd) of [3H]RX821002 was
essentially identical in the ciliary body, iris, and
RPE–choriocapillaris but slightly lower (higher
Kd) in the neurosensory retina (Table 1) .
The Kd values for these ocular tissues are
similar to those obtained under identical binding conditions for the
cloned 2A subtype (0.25 nM), but lower than those for
the 2B and 2C subtypes (0.89 and 0.58 nM,
respectively),22
indicating that the 2A may
be the major subtype in these tissues.
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2 adrenergic receptor subtypes are present
in human ocular tissues. In the ciliary body, various adrenergic agents
inhibited [3H]RX821002 binding with the expected rank
order for an 2A receptor (Fig. 2)
. With the exception of the agonist norepinephrine, none of the slope
factors (pseudo Hill coefficients) was significantly less than 1.0,
indicating the presence of a single major receptor subtype.
Oxymetazoline was approximately 300 times more potent than prazosin in
inhibiting [3H]RX821002 binding (Table 2)
, similar to the 100-fold difference found for the cloned
2A subtype and much different from the prazosin and
oxymetazoline ratios of 0.04 and 0.35 found for the cloned
2B and 2C subtypes,
respectively.26
The affinities of the antagonists used for
the 2B and 2C subtypes relative to the
2A subtype are shown in Table 3
. The large range of relative affinities (230–0.02) indicates that this
set of agents can easily differentiate the 2A subtype
from the 2B and 2C subtypes. The
Ki values determined for the iris,
RPE–choriocapillaris, and neurosensory retina were very similar to
those of the ciliary body (Table 2)
, indicating that the
2A subtype is also the predominant 2
adrenergic receptor subtype in these tissues.
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2A subtype (r = 0.97) but
poorly with the 2B (r = 0.22) and
the 2C (r = 0.50) subtypes. Similar
results were obtained for the other three human ocular tissues (Table 4)
.
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2A subtype
is the predominant 2 adrenergic receptor subtype in
human ocular tissues, the possibility of a low density of an additional
2 subtype cannot be excluded. In the porcine
neurosensory retina, for example, 85% of the receptors are
2A, but 15% are 2C.18
Furthermore, the 2C subtype is the main 2
receptor in a human retinoblastoma cell line (Y79).27
These observations prompted a more careful evaluation of the data. The
Kd value for [3H]RX821002 was
somewhat higher in the neurosensory retina than in the other tissues
(Table 1) . Similarly, in the neurosensory retina the slope factors for
many of the antagonists were less than 1.0. Spiroxatrine has a 40-fold
higher affinity for the human 2C subtype, compared with
the 2A, and thus is a good antagonist for detecting a
minor amount of 2C in the presence of the
2A subtype. If a measurable amount of 2C
were present in the neurosensory retina, then the spiroxatrine
inhibition data should fit a two-site model better than a one-site
model. In four of six inhibition experiments in the neurosensory
retina, the data fit a two-site model significantly better than a
one-site model (P < 0.05). For the four
experiments that modeled better as two sites, the higher affinity site
had a median effective concentration (EC50) of
1.4 nM and accounted for 56% of the receptors, whereas the lower
affinity site had an EC50 of 72 nM. When the data for all
six experiments were combined and fit as a single curve, similar
results were obtained (Fig. 5)
. Thus, it appears likely that the human neurosensory retina contains a
significant amount of both the 2A and 2C
subtypes.
|
Alpha-2 adrenergic agents, such as brimonidine and apraclonidine,
effectively lower intraocular pressure, although the mechanisms
involved are not yet well understood. Three presumed sites of action
for these agonists are the ciliary nonpigmented epithelium (reduction
of aqueous humor production), ciliary muscle (increase in uveoscleral
outflow facility), and the trabecular meshwork (increase in trabecular
outflow). In addition, in some species a central site of action for
2 agonists is also probable. Brimonidine and
apraclonidine are equally efficacious in decreasing aqueous humor
production,28
presumably by acting on the nonpigmented
epithelium of the ciliary body. However, brimonidine7
and
oxymetazoline29
also appear to increase uveoscleral
outflow, whereas apraclonidine increases outflow through the trabecular
meshwork.6
One potential explanation for these differences
is that the agonists have differential potencies at the three
2 adrenergic receptor subtypes, and that these subtypes
are differentially located in the relevant ocular tissues.
Several techniques have been used to define the localization of
2 adrenergic receptors in the human eye.
Autoradiographic studies found high levels of 2
adrenergic receptors in the iris epithelium and ciliary epithelium, as
well as in the ciliary muscle, retina, and RPE.30
In these
studies, the total pool of 2 receptors was visualized,
but the individual receptor subtypes were not considered. In the human
ciliary body, immunofluorescence labeling indicates the presence of the
2B and 2C subtypes, but not the
2A subtype.20
In contrast to these
immunofluorescence results, our radioligand-binding data indicate that
the 2A is the main, if not the only, subtype present in
the human ciliary body, iris, and RPE–choriocapillaris. The
neurosensory retina appears to contain mostly 2A and
perhaps some 2C. This conclusion is based on a
comparison of Ki values in the ocular
tissues with previous data from our laboratory with the cloned human
subtypes expressed in COS cells.26
Our conclusion that the
2A is the major subtype in human ocular tissues is
consistent with radioligand-binding studies in the cow, rabbit, and
pig, which also identify the 2A subtype as the main
ocular subtype.16
18
19
In contrast to our results from the radioligand-binding technique are
the results obtained using PCR and immunofluorescence techniques. Two
studies have used PCR to determine the absence or presence of mRNA
encoding the three 2 adrenergic receptor subtypes in the
human eye. In a transformed cell line of human nonpigmented epithelium,
PCR studies indicate the presence of mRNA for the 2B and
2C subtypes, but not the
2A.21
In a second, similar study published
thus far only in abstract form, the PCR technique indicated the
presence of only the 2B subtype in a transformed cell
line of human nonpigmented epithelium and in the human ciliary
body.21
Both studies also examined rabbit iris–ciliary
body. The former study found evidence for mRNA for all three subtypes,
whereas the latter study found only the 2A and
2B subtypes. The single study using the
immunofluorescence technique suggested the presence of the
2B and 2C subtypes, but not the
2A, in the human ciliary body.20
By
contrast all three subtypes were found in the rabbit iris–ciliary
body. Thus, although these two techniques appear to differ on exactly
which subtypes are present in human and rabbit ocular tissues, they
agree that the 2A subtype is absent in human tissues
studied. This conclusion is not supported by the results of the
radioligand-binding studies reported here.
It is of interest that the immunofluorescence technique identifies all
three subtypes in the rabbit ciliary body,20
whereas the
radioligand-binding approach finds only the 2A
subtype.19
Because immunofluorescence is not a
quantitative approach, it may be that the other two subtypes are
present in such low concentrations in the rabbit ciliary body that they
are not detected by the radioligand-binding technique, or that they are
not functional (i.e., unable to bind ligand) proteins. That there was
no immunofluorescence detection of the 2A subtype in the
human ciliary body is of concern, because this is the major subtype
detected by the radioligand technique in all four species investigated
to date: human, cow, pig, and rabbit. Furthermore, the cultured human
trabecular meshwork cells express only the 2A
subtype.31
As Huang et al.20
point out, the
2A subtype may well exist in the human eye but was not
detected in their immunofluorescence experiments, perhaps because the
antibody used was not sufficiently sensitive. However, the PCR studies
should have detected the mRNA for the 2A subtype if it
were present. Similarly, it is possible that the 2B and
2C subtypes are present at low density in the human
ciliary body, and thus were not detected by the radioligand technique
because of the presence of a much higher density of the
2A subtype. Another potential resolution to the
difference in conclusions reached by the two techniques is one of
receptor subtype localization, because the radioligand-binding
technique detects the binding in the tissue as a whole, whereas the
immunofluorescence experiments detect receptors only in a small area.
Thus, the 2A subtype could be the major subtype in the
ciliary body but did not happen to be in the specific place studied in
the immunofluorescence experiments. Conversely, the 2B
and 2C subtypes may be localized to the specific places
studied in the immunofluorescence experiments, but not sufficiently
widely distributed to be detected by the radioligand-binding technique.
Based on the data in Figure 5
, it appears probable that some
2C (or possibly 2B) receptors are present
in the neurosensory retina. A firm conclusion cannot be drawn, however,
because the data were significantly better fit by a two-site model in
only four of the six individual experiments. In addition, the
IC50 values for spiroxatrine derived from Figure 5
(3.7
µM and 104 µM) do not agree well with the
Ki values determined for the human clones
(5.5, 0.19, 0.13 µM) for the 2A, 2B,
and 2C subtypes, respectively.26
The low
density of 2 adrenergic receptors in the neurosensory
retina and the limited availability of human tissue make it difficult
to resolve this issue clearly.
The density of 2 adrenergic receptors has now been
determined in one or more ocular tissues in four species, as is
summarized in Fig. 6
. It is remarkable that the relative density in the four tissues is
different in each species and that the highest density for each tissue
is found in a different species. For example, in the cow, the receptor
density in the neurosensory retina is approximately 100 times higher
than in the human. The significance of this marked species variability
is unknown at the present time. Furthermore, it must be emphasized that
receptor density does not necessarily relate to either its
physiological or pharmacologic importance.
|
2 receptor density is relatively constant in the three
species that have been examined to date: human, 63 femtomoles/mg
protein (David Bylund, unpublished); cow, 71 femtomoles/mg
protein35
; and rat, 69 femtomoles/mg
protein.36
Some 2 adrenergic agents, including those used in
the treatment of glaucoma, also bind to the nonadrenergic imidazoline
sites. The I1 imidazoline site may have a role in
regulating blood pressure,37
but its role, if any, in
regulating intraocular pressure is unknown.38
39
Our
studies do not address this issue because the radioligand that we chose
([3HRX821002) has a low affinity for imidazoline
sites40
and thus would not bind to those sites under the
conditions of our assay. In addition, norepinephrine, which does not
bind to imidazoline sites, was used to define nonspecific binding in
our studies, thereby eliminating any contribution of imidazoline sites
to our data.
Alpha-2 adrenergic agonists are increasingly used in glaucoma therapy.
There is some evidence to suggest that these agents may have some
direct neuroprotective effect on the optic nerve in addition to the
protective effect of reduced intraocular pressure.41
42
However, long-term studies are needed to evaluate the extent to which
2 adrenergic agents preserve visual function. Attempts
to design new 2 agents with increased specificity and
thus fewer side effects will be strengthened by a better understanding
of the 2 adrenergic receptor subtype(s) mediating the
ocular hypotensive effects of these agents.
Acknowledgements
The authors thank Ronald J. Walkenbach and the Missouri Lions Eye Tissue Bank, Columbia, for the human eyes used in this study and Laurie J. Iversen for excellent technical assistance.
Footnotes
Supported by a seed grant from the University of Nebraska Medical Center and a grant from Allergan, Inc.
Submitted for publication January 20, 1999; revised March 26, 1999; accepted April 20, 1999.
Proprietary interest category: C5.
Corresponding author: David B. Bylund, Department of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, NE 68198-6260. E-mail: dbylund@unmc.edu
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2-adrenoceptors of the human retinoblastoma cell line (Y79) may represent an additional example of the 2C-adrenoceptor Br J Pharmacol 107,222-225[Medline][Order article via Infotrieve]
2A adrenergic receptors Invest Ophthalmol Vis Sci 37,2426-24332d-adrenergic receptors in bovine pineal gland Mol Pharmacol 40,235-241[Abstract]
2-adrenergic agonists induce basic fibroblast growth factor expression in photoreceptors in vivo and ameliorate light damage J Neurosci 16,5986-5992 2-adrenoreceptor agonists are neuroprotective in a rat model of optic nerve degeneration Invest Ophthalmol Vis Sci 40,65-73This article has been cited by other articles:
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  M. Saylor, L. K. McLoon, A. R. Harrison, and M. S. Lee Experimental and Clinical Evidence for Brimonidine as an Optic Nerve and Retinal Neuroprotective Agent: An Evidence-Based Review Arch Ophthalmol, April 1, 2009; 127(4): 402 - 406. [Abstract] [Full Text] [PDF]
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 M. Aihara, J. D. Lindsey, and R. N. Weinreb Effect on Diurnal Intraocular Pressure Variation of Eliminating the {alpha}-2 Adrenergic Receptor Subtypes in the Mouse Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 929 - 933. [Abstract] [Full Text] [PDF]
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 R. H. Rosa Jr., T. W. Hein, Z. Yuan, W. Xu, M. I. Pechal, R. L. Geraets, J. M. Newman, and L. Kuo Brimonidine evokes heterogeneous vasomotor response of retinal arterioles: diminished nitric oxide-mediated vasodilation when size goes small Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H231 - H238. [Abstract] [Full Text] [PDF]
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A. E. de Salamanca, K. F. Siemasko, Y. Diebold, M. Calonge, J. Gao, M. Juarez-Campo, and M. E. Stern Expression of Muscarinic and Adrenergic Receptors in Normal Human Conjunctival Epithelium Invest. Ophthalmol. Vis. Sci., February 1, 2005; 46(2): 504 - 513. [Abstract] [Full Text] [PDF]
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D W Evans, S L Hosking, D Gherghel, and J D Bartlett Contrast sensitivity improves after brimonidine therapy in primary open angle glaucoma: a case for neuroprotection Br. J. Ophthalmol., December 1, 2003; 87(12): 1463 - 1465. [Abstract] [Full Text] [PDF]
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 S. Guimaraes and D. Moura Vascular Adrenoceptors: An Update Pharmacol. Rev., June 1, 2001; 53(2): 319 - 356. [Abstract] [Full Text] [PDF]
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