May 1983
Vol. 24/5
Investigative Ophthalmology
b Visual Science
A Journal of Dosic and Clinical Research
Articles
Changes in Responsiveness of the /?-Adrenergic and
Serotonergic Pathways of the
Rabbit Corneal Epithelium
Arthur H. Neufeld, Sally E. Ledgord, and Barbara K. Yoza
Adrenergic agonists stimulate the synthesis of cyclic AMP by incubated rabbit corneas with the
following order of potency: isoproterenol > epinephrine > norepinephrine. These agonists have the
same order of potency when displacing the specific, /3-adrenergic radioligand, 3H-dihydroalprenolol,
from /8-adrenergic receptors on membranes prepared from corneal epithelium. At another locus,
serotonin stimulates cyclic AMP synthesis. Inhibition of stimulation in vitro by lysergic acid diethylamide, methysergide, cyproheptadine, and spiroperidol demonstrates the specificity of this pathway
for serotonin. Topical epinephrine causes subsensitivity or decreased responsiveness of the 0-adrenergic pathway. There is loss of approximately half the /?-adrenergic receptors from the cornea and
a similar loss of epinephrine-stimulated cyclic AMP synthesis, both of which return to control levels
in 96 hrs. There is no change in affinity for catecholamines and no loss of responsiveness to prostaglandin E2 or serotonin. Pretreatment with nialamide or subsequent treatment with additional epinephrine does not cause further loss of responsiveness. Supersensitivity or increased responsiveness
of this pathway occurs following superior cervical ganglionectomy. Topical serotonin causes decreased
responsiveness of the serotonergic pathway. When potentiated by nialamide, serotonin causes almost
complete loss of serotonin-stimulated cyclic AMP synthesis for 24-48 hrs. There is no loss of responsiveness to epinephrine. Increased responsiveness of this pathway does not occur following superior cervical ganglionectomy. The authors conclude that the corneal epithelium has both /?2-adrenergic and serotonin-2 pathways, and each pathway exhibits altered responsiveness by similar mechanisms. In response to exogenous or endogenous stimulation, the /8-adrenergic responsive cells and
the serotonergic responsive cells apparently regulate the total number of pathway-specific receptors
on their surfaces. Furthermore, the authors postulate that two populations of 0-adrenergic responsive
cells exist; those on the apical surface of the epithelium that respond to catecholamine in the tears
and those near the basal surface that respond to neuronal catecholamine. Invest Ophthalmol Vis Sci
24:527-534, 1983
mediated by cyclic AMP include relaxation of bronchial, intestinal and uterine smooth muscle, liver glycogenolysis, and fat cell lipolysis.2 In the eye, cyclic
AMP affects corneal epithelial wound closure3 and
may mediate cellular mechanisms regulating intraocular pressure.4"8
In the corneal epithelium, catecholamines such as
epinephrine and norepinephrine act at 0-adrenergic
receptors to stimulate, via cyclic AMP, chloride transport9"11 and to influence mitotic rate.12 At other receptors in the corneal epithelium, serotonin also stimulates the synthesis of cyclic AMP13 and activates
chloride transport,14 but in a manner that suggests a
spatial separation of serotonergic and /3-adrenergic
receptors.
Receptors on the cell surface have an important
role in cellular response pathways. For example, the
binding of catecholamines to /3-adrenergic receptors
on the target cell membrane leads to activation of
adenylate cyclase, the enzyme that synthesizes the
intracellular second messenger, cyclic AMP.1 Physiologic responses stimulated by catecholamines and
From the Eye Research Institute of Retina Foundation and the
Department of Ophthalmology, Harvard Medical School, Boston,
Massachusetts.
Supported in part by USPHS Grants EY-02360 and EY-02367.
Submitted for publication May 27, 1982.
Reprint request: Arthur H. Neufeld, Eye Research Institute, 20
Staniford Street, Boston, MA 02114.
0146-0404/83/0500/527/$ 1.20 © Association for Research in Vision and Ophthalmology
527
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528
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1983
/3-adrenergic receptors are defined by their relative
affinities for certain agonists, usually: isoproterenol
> epinephrine ^ norepinephrine.1 Pharmacologic
evidence indicates that there are two types of 0-adrenergic receptors. j8radrenergic receptors, found in
cardiac and adipose tissues, have equal affinities for
epinephrine and norepinephrine; 02-adrenergic receptors, found in pulmonary and vascular smooth
muscle, have a greater affinity for epinephrine than
for norepinephrine. Biochemical studies have demonstrated that the order of potency of catecholamines
to stimulate cyclic AMP synthesis parallels the affinities of catecholamines for j8 r and /32-adrenergic receptors.
Serotonergic receptors are also defined by their relative affinities for certain drugs. In the central nervous
system, two types of receptors have been identified
that have either high or low affinity for serotonin.15"17 We have previously reported that the corneal
epithelium has a low affinity serotonergic pathway
that is mediated by cyclic AMP.13
Studies in several tissues have demonstrated subsensitivity or loss of responsiveness due to down regulation of the /3-adrenergic pathway: catecholamineinduced loss of /3-adrenergic receptors accompanied
by decreased catecholamine-stimulated cyclic AMP
synthesis.1 In rabbit corneas, repeated topical epinephrine treatment decreases 0-adrenergic receptor
density and depresses epinephrine-stimulated cyclic
AMP synthesis and chloride transport by the epithelium.1819
Subsensitivity of the serotonergic pathway has been
reported for brain tissue. Like the change in density
that occurs with /3-adrenergic receptors, decreased
responsiveness to serotonin may also be due to a decreased number of receptors for the transmitter.20
In the experiments to be presented, we have characterized the j8-adrenergic and serotonergic pathways
in the rabbit corneal epithelium and described the
changes in responsiveness that occur following various treatments.
Materials and Methods
Treatment of Animals .
Male, albino, New Zealand rabbits, weighing 2-3
kg, were killed with an intravenous overdose of sodium pentobarbital.
Two drops of 40 mg/ml 1-epinephrine, d-bitartrate
or 10 mg/ml serotonin in isotonic saline were applied
to one eye of each rabbit, and animals were killed at
various intervals after treatment. In some experiments, a second treatment was given 2 hrs after the
initial dose. In other experiments, eyes were pretreated topically with two drops of 1 mg/ml nialamide
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Vol. 24
without further treatment or treated, 5 min later, with
topical epinephrine or serotonin as above.
Some animals had a unilateral, superior cervical
ganglionectomy at least 30 days before use.
Preparation of Membranes
Epithelia from two or more fresh (or frozen) rabbit
corneas were scraped off with a Bard-Parker no. 10
surgical blade and placed in 0.5-1.0 ml of cold buffer
containing 50 raM Tris, 10 mM MgCl2, pH 7.55.
Epithelia were homogenized in a glass/Teflon homogenizer, and the protein concentration was determined by the method of Lowry et al.21 Epithelial homogenates were either used for membrane assays the
same day or were frozen at —20 C.
Assay for #-adrenergic Receptors
/3-adrenergic receptors were assayed by a modification22 of the method of Mukherjee et al.23 Two
hundred micrograms of protein homogenate were incubated in 200 ft\ of buffer containing: 50 mM Tris,
15 mM MgCl2, 0.1 mM 3'5'-dihydroxymethylpropiophenone and 0.013 mM clorgyline, pH 8.1, for 10
min in the presence of 26 nM 3H-dihydroalprenolol
(3H-DHA) at 37 C. Varying concentrations of isoproterenol, epinephrine, and norepinephrine were
added to the incubation media to demonstrate specificity. Specific binding was defined as the difference
between the amount of 3H-DHA bound in the presence (nonspecific binding) and absence (total binding)
of 10 MM propranolol.
In Vitro Cyclic AMP Synthesis
Full-thickness corneal pieces were incubated in
vitro in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and assayed for cyclic AMP levels
as previously described.24 Indomethacin, 0.1 mg/ml,
was added to the incubation buffers, and in some
experiments 0.01 mM nialamide was present. Corneas were challenged with drugs at the final concentration indicated.
Materials
3
H-DHA (49.1 Ci/mmol) was purchased from New
England Nuclear; components for the radioimmunoassay for cyclic AMP were from Collaborative Research; 1-epinephrine, d-bitartrate, 1-isoproterenol, dbitartrate, 1-arterenol, d-bitartrate, dl-propranolol HC1,
indomethacin, serotonin, nialamide, and IBMX were
from Sigma; PGE2 and 3'5'-dihydroxymethylpropiophenone from Upjohn; clorgyline from May and
Baker; timolol and cyproheptadine from Merck; LSD
529
0-ADRENERGIC AND SEROTONERGIC PATHWAYS / Neufeld er ol.
No. 5
100i
O
10
10
[AGONIST]
Fig. 1. Stimulation of cyclic AMP synthesis by adrenergic agonists. Percent maximum was derived by comparing all agonist stimulated values minus basal values to the stimulated value in the
presence of 10 nM isoproterenol minus the basal value. ISOP
(O), isoproterenol; EPI (•), epinephrine; NOREPI (X), norepinephrine. All values are the mean of at least six determinations.
and methysergide maleate from Sandoz; and spiro-
peridol from Janssen. Frozen eyes were from PelFreez Biologicals. All other compounds used were
reagent grade.
Results
/8-adrenergic Pathway
Figure 1 illustrates the ability of adrenergic agonists
to stimulate the synthesis of cyclic AMP in the cornea. The order of potency of these agonists, isoproterenol > epinephrine > norepinephrine, indicates
that the corneal epithelium is predominantly a /32adrenergic responsive pathway. Further proof is provided in Figure 2, which shows the ability of the adrenergic agonists to displace the specific, 0-adrenergic
radioligand, 3H-DHA, from binding sites on corneal
epithelial membranes. The order of potency for displacement, isoproterenol > epinephrine > norepinephrine, is the same as that for stimulating the synthesis of cyclic AMP.
[AGONIST]
Fig. 2. Displacement of 3H-DHA binding by adrenergic agonists.
% Maximum was derived by comparing all values for specific binding in the presence of agonists with the amount of specific binding
as defined by the absence and presence of 10 /xM propranolol. All
values are the mean of at least four determinations.
Decreased Responsiveness
Table 2 demonstrates that topical, unilateral treatment with 2% epinephrine causes loss of /3-adrenergic
responsiveness, as measured 3 hr later by epinephrine-stimulated cyclic AMP synthesis in vitro, in the
treated eye compared to the untreated control eye.
Decreased responsiveness following topical treatment
with epinephrine is not potentiated by topical pretreatment with nialamide. Table 2 also demonstrates
that the subsensitivity that occurs following a single
administration of topical epinephrine is maximal;
additional administration of topical epinephrine, 2
hrs after the initial dose, does not cause a further loss
of responsiveness of the /3-adrenergic pathway.
Table 1. Inhibition of serotonin-stimulated cyclic
AMP synthesis
pmol cyclic AMP/mg
prot/15 min
Serotonergic Pathway
Table 1 demonstrates the specificity of serotoninstimulated synthesis of cyclic AMP. At 10 txM, specific, serotonergic antagonists, such as LSD, methysergide, cyproheptadine, and to a lesser extent, spiroperidol, block stimulation of the synthesis of cyclic
AMP by serotonin. The /3-adrenergic antagonist, timolol is not effective.
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10~3M
10
Basal
Serotonin (100 pM)
Epinephrine (10 MM)
Serotonin + LSD (10 ^M)
Serotonin + methysurgide (10 nM)
Serotonin + cyproheptadine (10 j*M)
Serotonin + spiroperidol (10 fiM)
Serotonin + timolol (10 pM)
* Mean ± SEM (number of determinations = 6).
7.1 ±0.8*
24.0 ± 2.0
130.0 ± 8.0
7.6 ± 0.9
6.5 ± 0.8
8.0 ± 1.3
11.6 ± 1.8
24.5 ± 1.3
530
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / May 1983
Table 2. Epinephrine* stimulated cyclic AMP
synthesis, in vitro
pmol cyclic AMP/mg
prot/15 min
Treatment, in vivo
None
Topical epinephrine
Topical nialamide and epinephrine
Topical epinephrine (twice: at 0 and 2 hr)
142 ±
66 ±
85 ±
73 ±
9 (12)f
8 (10)
8 (12)
8 (4)
t Mean ± SEM (number of eyes).
Table 3 shows that topical treatment with serotonin, when combined with topical pretreatment with
nialamide, causes loss of serotonergic responsiveness,
as measured 3 hr later by serotonin-stimulated cyclic
AMP synthesis in vitro. Decreased responsiveness of
the serotonergic pathway is potentiated by nialamide
pretreatment; topical treatment with nialamide alone
has no effect (data not shown).
Figure 3 illustrates the time course of subsensitivity
of the 0-adrenergic pathway. Loss of responsiveness
occurs within 3 hrs after topical treatment, at which
time the ability of the pathway to respond to catecholamine has been reduced to approximately 50%.
Responsiveness of the 0-adrenergic pathway returns,
in a linear manner, over the subsequent 96 hrs after
topical treatment with epinephrine. The time course
of changes in /3-adrenergic receptor density, as measured by specific 3H-DHA binding, indicates that
down regulation of receptor density exactly parallels
the return of epinephrine-stimulated cyclic AMP synthesis. At 0 time, we measured 52 fmol 3H-DHA specifically bound/mg corneal epithelial membrane protein, which decreased to 29 fmol 3H-DHA/mg protein
3 hr after topical epinephrine, remained decreased
(71% of control at 48 hr), and returned to control
values at 96 hrs.
Figure 3 also illustrates the time course of subsensitivity of the serotonergic pathway. This time course
is quite different than that of the 0-adrenergic pathway. Within 3 hrs after topical treatment with nialamide and serotonin, almost all responsiveness is
lost. Decreased responsiveness persists for approximately 24 hrs and then returns to control levels between 24 and 48 hrs after topical treatment with serotonin.
Table 3. Serotonin* stimulated cyclic AMP
synthesis, in vitro
Treatment, in vivo
pmol cyclic AMP/mg
prot/15 min
None
Topical serotonin
Topical nialamide and serotonin
25 ± 2 (8)t
20 ± 3 (14)
14 ± 2 (22)
• 100 tiU.
t Mean ± SEM (number of eyes).
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Vol. 24
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Fig. 3. Time course of decreased responsiveness following topical
treatment with epinephrine (O) or serotonin (•). Epinephrine (10
nM) or serotonin (100 ^M) stimulated cyclic AMP synthesis was
compared in treated and contralateral, untreated eyes. All values
are the mean of at least four determinations.
Table 4 demonstrates that epinephrine-induced
subsensitivity of the /3-adrenergic pathway is specific.
In response to a single administration of topical epinephrine, decreased responsiveness does not occur to
either prostaglandin E2 or serotonin. Similarly, subsensitivity that occurs in response to topical serotonin does not affect responsiveness to epinephrine
(Table 4).
Figure 4 illustrates that following topical epinephrine there is a loss of maximal responsiveness of the
/3-adrenergic pathway with no change in the apparent,
half-maximal concentration of epinephrine for stimulating cyclic AMP synthesis. Similarly, the maximal
responses to isoproterenol and norepinephrine decrease following topical treatment with epinephrine
with no apparent change in half-maximal concentrations for stimulating cyclic AMP synthesis (data not
shown).
Figure 5 illustrates that following topical treatment
with nialamide and serotonin, there is a marked loss
of maximal responsiveness of the serotonergic pathway for stimulating cyclic AMP synthesis. There may
also be a change in affinity for serotonin of the pathway but this is not possible to determine from this
data.
Increased Responsiveness
Figure 6 demonstrates that supersensitivity occurs
in the /3-adrenergic pathway of the cornea following
removal of the ipsilateral, superior cervical ganglion.
0-ADRENERGIC AND SEROTONERGIC PATHWAYS / Neufeld er ol.
No. 5
531
Table 4. Cyclic AMP synthesis, in vitro, 3 hr after in vivo treatment with topical drug
pmol cyclic AMP/mg prot/15 min
Control
Topical epinephrine
Topical serotonin
Basal
10 fiM epinephrine
10 \iM prostaglandin E2
100 nM serotonin
16 ± 2(12)*
21 ±5(12)
10 ± 1 (12)
142 ± (12)
64 ± 6(6)
123 ± 10(6)
64 ± 6 (12)
70 ± 7 (6)
not determined
30 ±2(4)
29 ±4(4)
12 ± 1 (12)
Mean ± SEM (number of eyes).
In vitro, epinephrine stimulates the synthesis of more
cyclic AMP in corneas from denervated eyes compared to corneas from contralateral, innervated eyes
(P > 0.02). As shown in Figure 7, this increased responsiveness is apparently due to an increased number of /3-adrenergic receptors in corneas of the denervated eyes compared to the innervated eyes (P
> 0.01). No change in affinity for catecholamine occurs in the /3-adrenergic pathway of the cornea following adrenergic denervation (data not shown). Figure 6 also demonstrates that topical epinephrine
causes subsensitivity in corneas that are supersensitive following denervation.
Figure 8 demonstrates that supersensitivity of the
serotonergic pathway does not occur following removal of the superior cervical ganglion. As determined measuring serotonin-stimulated cyclic AMP
synthesis in vitro, there is no apparent increase in
total responsiveness nor any change in the half-maximal concentration for activation of this pathway by
serotonin following denervation.
Discussion
The relative abilities of the adrenergic agonists, isoproterenol, epinephrine, and norepinephrine, to stimulate the synthesis of cyclic AMP and to displace the
specific radioligand, 3H-DHA, indicate that the corneal epithelium contains /?2-adrenergic receptors. We
cannot rule out that this tissue may also contain a
significant number of /31 -adrenergic receptors. However, considering the relative separation between the
epinephrine and norepinephrine curves, both for
cyclic AMP synthesis and radioligand binding and
the homogeneity of cell type, we conclude that the
predominant form of the receptor is fo-adrenergic.
The corneal epithelium also contains a specific serotonergic pathway that, independently of the /3-adrenergic pathway, stimulates the synthesis of cyclic
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[SEROTONIN]
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Fig. 4. Stimulation of cyclic AMP synthesis by epinephrine in
vitro 3 hrs after topical treatment of one eye with epinephrine
(TOP EPI, O) compared to the contralateral eye (CONT, • ) . Percent maximum was derived by comparing all epinephrine-stimulated values minus basal values to the stimulated value in the untreated eyes in the presence of 10 nM epinephrine minus the basal
value. All values are the mean of at least six determinations.
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Fig. 5. Stimulation of cyclic AMP synthesis by serotonin in vitro
3 hrs after topical treatment of one eye with serotonin (TOP SER,
O) compared to the contralateral eye (CONT, • ) . Both eyes were
treated with nialamide. Percent maximum was derived by comparing all serotonin-stimulated values minus basal values to the
stimulated value in the untreated eyes in the presence of 100 nM
serotonin minus the basal value. All values are the mean of at least
six determinations.
532
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TOPICAL EPINEPHRINE
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Vol. 24
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / May 1983
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10±
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o
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BASAL
-EPI-STIMULATED.IN VITRO-
Fig. 6. Changes in the /3-adrenergic pathway following ipsilateral
superior cervical ganglionectomy (shaded) compared to the contralateral, innervated eye (unshaded). Topical epinephrine animals
were treated in vivo 3 hrs before excision of the corneas. Epinephrine (10 juM)-stimulated cyclic AMP synthesis was measured
in vitro. All values are the mean ± SEM of six animals.
200 T
150-o
Q
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X
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X
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/3-ADRENERGIC RECEPTORS
Fig. 7. Changes in the /3-adrenergic pathway following ipsilateral,
superior cervical ganglionectomy (shaded) compared to the contralateral, innervated eye (unshaded). /3-adrenergic receptor density
is indicated by the specific binding of 3H-DHA to membranes. All
values are the mean ± SEM of six determinations.
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10 - 8
10 - 7
10 -6
10 - 5
M
[SEROTONIN]
Fig. 8. Stimulation of cyclic AMP synthesis by serotonin in vitro
following unilateral, superior cervical ganglionectomy (O) compared to the contralateral, innervated eye (•). All values are the
mean of six determinations.
AMP. Considering the low affinity for serotonin, as
measured by cyclic AMP synthesis 13 and displacement of the specific radioligand, 3H-LSD,* the serotonergic receptors of this pathway appear similar to
the serotonin-2 type described for the central nervous
system.2526 In the cornea, we find no evidence for the
presence of serotonin-1 receptors, which in the brain
have a high affinity for serotonin and are also linked
to the synthesis of cyclic AMP.
Following topical administration of epinephrine,
decreased /?-adrenergic responsiveness of the corneal
epithelium occurs.1819 An approximately 50% loss of
/5-adrenergic receptor density is reflected in an approximately 50% loss of /3-adrenergic-stimulated cyclic
AMP synthesis. The subsensitivity is due to down
regulation of the receptor population and not to a
change in affinity for /3-adrenergic agonists.
The loss of about half of the 0-adrenergic responsiveness of the tissue is apparently the maximal loss
that can occur. Nialamide, the monoamine oxidase
inhibitor, which might effectively increase the concentration of epinephrine in the cornea by preventing
metabolic breakdown, does not potentiate the decreased responsiveness following topical epinephrine.
Similarly, a second dose of topical epinephrine (given
2 hr after the first dose) does not cause further loss
of responsiveness. Thus, the total number of /?-adrenergic receptors in the corneal epithelium cannot
decrease below a lower limit, even in the presence of
a higher concentration of exogenous epinephrine.
Activation of the /3-adrenergic pathway stimulates
C\~ transport by apical cells of the corneal epithelium.
* Villarreal VV and Neufeld AH. Unpublished results.
No. 5
/3-ADRENERGIC AND SEROTONERGIC PATHWAYS / Neufeld er ol.
The loss of approximately half of the /3-adrenergic
receptors following topical epinephrine is associated
with an essentially complete loss of the ability of catecholamines to stimulate chloride transport.19 Therefore, we conclude that approximately half of the
0-adrenergic receptors of the cornea are linked to
stimulation of chloride transport. These /3-adrenergic
receptors are located on the anterior membrane of
the apical cells and down regulation apparently leads
to complete loss of their activity.
Perhaps the remaining /3-adrenergic receptors are
located in the basal epithelium and associated with
some aspect of epithelial movement or proliferation.
These cells are probably responsive to norepinephrine
released from corneal adrenergic nerves, and, therefore, supersensitivity following denervation may be
more likely to occur in the /3-adrenergic pathway of
this layer. Thus, we hypothesize that the /3-adrenergic
pathway in the apical epithelium responds primarily
to catecholamines delivered via the tears and the /?adrenergic pathway in the basal epithelium responds
primarily to neuronal catecholamines.
Subsensitivity of the 0-adrenergic pathway persists
for a prolonged period after a single administration
of epinephrine. Although de novo synthesis of /?-adrenergic receptors in existing cells may occur over 96
hrs, the time course suggests that the return to control
levels of responsiveness may be due to the turnover
and replacement of apical cells by wing cells moving
anteriorly. Thus, once down regulated, the apical cells
in the corneal epithelium may not synthesize new
/3-adrenergic receptors.
Subsensitivity also occurs in the serotonergic pathway by a mechanism similar to that for the /3-adrenergic pathway. We hypothesize that down regulation
of serotonergic receptors occurs, but the low affinity
for serotonin in the radioligand binding assay does
not allow a direct test of this possibility. However,
certain differences in loss of adrenergic and serotonergic responsiveness are noteworthy. Topical serotonin causes an almost complete loss of responsiveness of the serotonergic pathway. Because serotonergic receptors are located deeper within the corneal
epithelium than the apical /5-adrenergic receptors,
nialamide pretreatment is necessary in vivo to potentiate serotonin-induced subsensitivity. Furthermore, the time course of the return of serotonergic
responsiveness to control values is unlike that of the
/3-adrenergic pathway.
Decreased responsiveness of a pathway only affects
that specific pathway and, therefore, must be due to
regulatory changes that alter molecular events before
the activation of adenylate cyclase. Subsensitivity in
the /?-adrenergic pathway does not alter the response
of the tissue to serotonin or prostaglandin E2. Similarly subsensitivity in the serotonergic pathway does
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533
not alter the ability of epinephrine to stimulate the
synthesis of cyclic AMP. Thus, the changes that occur
to cause loss of responsiveness must be at the receptor
level.
Many pathways that have a mechanism to decrease
responsiveness also have a mechanism to increase
responsiveness. The /3-adrenergic pathway in the corneal epithelium is such a system. Following surgical
removal of the superior cervical ganglion, when all
the adrenergic nerves to the ipsilateral eye have degenerated, we find a small increased responsiveness,
as measured by epinephrine-stimulated cyclic AMP
synthesis, and a small increase in the number of 0adrenergic receptors in the corneal epithelium. This
supersensitivity must be due to decreased adrenergic
stimulation following loss of the nerves. Thus, the
number of /3-adrenergic receptors in the corneal epithelium is a continuum, regulated in inverse proportion to humoral and neuronal adrenergic stimulation. Similar increases27 and decreases28'29 in adrenergic responsiveness in the iris-ciliary body have been
reported.
The serotonergic nerves of the cornea presumably
pass through the superior cervical ganglion.14 Nevertheless, supersensitivity of the serotonergic pathway
does not occur following removal of the ganglion.
Perhaps this pathway is already operating at maximal
responsiveness, or the serotonergic nerves do not
truly pass through the superior cervical ganglion.
In conclusion, the /32-adrenergic receptor pathway
and the serotonin-2 receptor pathway of the corneal
epithelium exhibit altered responsiveness depending
upon the level of specific stimulation. The mechanisms by which changes in responsiveness occur in
both pathways appear similar although the time
course of these changes and the location of the pathways in the tissue are quite different.
Key words: epinephrine, serotonin, /3-adrenergic, cornea,
rabbit, receptor, cyclic AMP, down regulation, denervation,
subsensitivity, supersensitivity
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Adrenergic Pharmacology. New York, Raven Press, 1978, pp.
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2. Sutherland EW: Studies on the mechanism of hormone action.
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3. Jumblatt MM, Fogle JA, and Neufeld AH: Cholera toxin stimulates adenosine 3',5'-monophosphate synthesis and epithelial
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534
INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / May 1983
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14. KJyce SD, Palkama KA, HSrkOnen M, Marshall WS, Huhtaniitty S, Mann KP, and Neufeld AH: Neural serotonin stimulates chloride transport in the rabbit corneal epithelium. Invest Ophthalmol Vis Sci 23:181, 1982.
15. Peroutka SJ and Snyder SH: Multiple serotonin receptors:
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