Departments of 1 Ophthalmology and 2 Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029
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ABSTRACT |
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The effects of serotonin
[5-hydroxytryptamine (5-HT)] on the transepithelial electrical
properties of the short-circuited rabbit conjunctiva were examined.
With this epithelium, the short-circuit current
(Isc) measures Cl secretion plus
an amiloride-resistant Na+ absorptive process. Apical
addition of 5-HT (10 µM) elicited a prompt Isc
reduction from 14.2 ± 1.2 to 10.9 ± 1.2 µA/cm2 and increased transepithelial resistance from
0.89 ± 0.05 to 1.03 ± 0.06 k
· cm2
(means ± SE, n = 21, P < 0.05).
Similar changes were obtained with conjunctivae bathed without
Na+ in the apical bath, as well as with conjunctivae
preexposed to bumetanide with the Cl
-dependent
Isc sustained by the parallel activities of
basolateral Na+/H+ and
Cl
/HCO
exchangers. In contrast, the
5-HT-evoked effects were attenuated by the absence of Cl
(
Isc =
0.5 ± 0.2, n = 5), suggesting that reduced Cl
conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated
conjunctiva and in the presence of a transepithelial K+
gradient, 5-HT addition reduced K+ diffusion across the
preparation by 13% and increased transepithelial resistance by 4%
(n = 6, P < 0.05), indicating that an
inhibition in K+ conductance(s) was also detectable.
Significant electrical responses also occurred under physiological
conditions when 5-HT was introduced to epithelia pretreated with
adrenergic agonists or protein kinase C, phospholipase C,
phosphodiesterase, or adenylyl cyclase inhibitors or after perturbation
of Ca2+ homeostasis. Briefly, the conjunctiva harbors the
only known Cl
-secreting epithelium in which 5-HT evokes
Cl
transport inhibition; receptor subtype and signal
transduction mechanism were not determined.
electrolyte transport; Ussing chamber; short-circuit current; serotonin receptors; chloride secretagogue
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INTRODUCTION |
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THE CONJUNCTIVA IS A
THIN, transparent, mucus-secreting, vascularized epithelium that
lines the inner surface of the eyelids (the palpebral conjunctiva) and
covers the anterior sclera of the ocular globe (the bulbar
conjunctiva). This epithelium is embryologically related to, and
anatomically continuous with, that of the upper airway. It is
stratified, with variations in cell layers from the tarsal portion of
the eyelid to the corneoscleral junction (45), and
manifests, as determined with rabbit specimens (20, 33),
electrical features characteristic of "tight epithelia," i.e., a
relatively high transepithelial resistance (Rt).
Its electrolyte transport properties are similar to those of the
mammalian intestinal epithelium (10) and the shark renal
proximal tubule (3), in which mechanisms for
Na+ absorption and Cl secretion coexist.
Identical to Cl-secreting epithelia, such as the
extensively characterized frog corneal epithelium (5), the
rabbit conjunctiva has a basolateral bumetanide-sensitive
Cl
uptake process (presumably
Na+-K+-2Cl
cotransport)
positioned in series with apical Cl
channels (20,
33). In addition, recently identified
Na+/H+ and
Cl
/HCO
exchangers that exist in
parallel in the basolateral membrane (41) can also mediate
Cl
uptake. The contribution of the acid-base transporters
to transepithelial Cl
secretion is variable and dependent
on individual rates of metabolic CO2 production, inasmuch
as the transepithelial electrical parameters do not require
extracellular HCO
(unpublished observations).
Oppositely directed, electrogenic Na+ reabsorption is amiloride insensitive (33) and occurs at the apical surface via Na+-dependent cotransporters such as those carrying glucose (14) and amino acids (21) in series with the basolaterally located Na+-K+ pump, an arrangement similar to that found in the intestine, kidney, and liver (32, 39, 44). Furthermore, nonselective cation channels were identified in whole cell patch clamping of freshly isolated conjunctival epithelial cells (42), and the possibility that such channels reside at the apical surface has been suggested (40).
Because of its frailty and the difficulty of the dissection needed to isolate the conjunctival epithelium intact, the above macroscopic electrolyte transport properties of this tissue, as measured in bicameral Ussing-type chambers, was characterized relatively recently (20). Hence, many fundamental aspects of the tissue have not been elucidated. The underlying rationale for characterizing conjunctival transport is a quest for elaborating the secretory functions of the epithelium under the premise that such efforts might have utility in ameliorating complications from dry-eye diseases. The surface area of the conjunctival epithelium is 9-fold larger in the rabbit and 17-fold larger in the human than that of the cornea (43). Thus, hypothetically, active transport by the conjunctiva with accompanying fluid secretion may contribute to a significant fraction of tear production, which is normally provided in healthy individuals by the lacrimal gland. On stimulation, the transepithelial conjunctival contribution could be greater.
Given that serotonin [5-hydroxytryptamine (5-HT)] is widely
recognized as a Cl secretagogue in epithelial tissues
(4, 7, 5, 12, 16, 19, 35-37, 49), it was posited a
priori that 5-HT might serve as a stimulator of conjunctival
Cl
transport as well. The presence of mast cells within
the conjunctival stroma (26) and the likelihood that these
cells may release 5-HT, as well as the facts that trace amounts of 5-HT
are found in tears (25, 46) and that 5-HT stimulates mucin
secretion from goblet cells within the epithelium (17),
suggested that the epithelium could harbor serotonergic receptors
potentially linked to the regulation of transepithelial transport.
However, it was unexpectedly found that 5-HT is an inhibitor of the
Cl
-dependent short-circuit current
(Isc) of the rabbit conjunctiva by a mechanism
that requires further clarification.
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METHODS |
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Adult albino rabbits of either sex weighing 2-3.6 kg were killed by CO2 asphyxiation. The bulbar-palpebral conjunctiva was dissected as a cylinder and cut longitudinally to convert it to a flat epithelium that was mounted as a partition between Ussing-type hemichambers, exposing 0.38 cm2 of cross-sectional area. The dissection was presented in detail previously (33) and was based on a procedure described by Kompella et al. (20).
The hemichambers included the necessary arrangements for electrical determinations and vigorous stirring. The transconjunctival potential difference was short-circuited, with the current needed to maintain 0 mV across the tissue (the Isc) continuously recorded. Transmural electrical resistance (Rt) was determined by measuring the amount of current necessary to offset the short-circuited condition by 2 or 3 mV for a few seconds.
In general, it was observed that conjunctival preparations from heavier
rabbits (3 kg) were less delicate and easier to handle and place in
the chambers, given the larger areas of tissue that could be readily
procured from such animals. The frailty of the preparation, however,
appeared to contribute to a spontaneous, gradual decline in
Rt that was commonly observed after a prolonged period in the chamber (10-30% reduction in
Rt in control preparations during 3-4 h of
observation). This decline is best explained as a loss in paracellular
resistance, since it occurred in the presence of a steady
Isc (unpublished data). Under the
short-circuited conditions, increases in paracellular ion movement do
not result in a net flow across this pathway, given the absence of a
potential difference across the epithelium and identical electrolyte
concentrations on each side of the preparation. Thus, although the
Isc measured net transcellular flow in
experiments with symmetrical solutions, conjunctival
Rt changes elicited by the additions of various
agents frequently underestimate changes in membrane resistance; i.e., because of the proportionally larger transcellular than paracellular resistance, large changes in transcellular resistance are seen as
smaller changes when Rt is measured.
Nevertheless, unless indicated otherwise, the Rt
changes given, although proportionally small in some sets of
experiments, were statistically significant as paired data
(P < 0.05) and reflect the
Rt values elicited by experimental agents at
the point of their introduction. Most illustrations of the electrical
changes were acquired by scanning the chart recordings with a page
scanner so that the background chart grids could be removed with
commercial software.
The bathing medium used during the dissection and bathing of the tissue in the chambers in most experiments was a modified Tyrode solution with the following composition (in mM): 1.8 calcium gluconate, 1.2 MgCl2, 4 KCl, 103 NaCl, 30 NaHCO3, 1 NaH2PO4, 5.7 glucose, 0.3 glutathione, and 10 sucrose. The pH of this solution when bubbled with 5% CO2-95% air was 7.5. It measured 280 mosmol/kgH2O.
In some experiments, gluconate was used as a Cl
substitute along with MgSO4 replacing MgCl2.
For an Na+-free medium, the sodium salts of HCl and
H3PO4 were replaced by their respective
N-methyl-D-glucamine (NMDG) salts and choline was used as the counter ion for bicarbonate. In experiments measuring K+ diffusion, a Cl
-free, high-K+
solution with low Na+ was used to bathe the apical aspect
of the conjunctiva, whereas the stromal side was bathed with a
Cl
-free medium containing 4 mM K+ and low
Na+. For the apical bath, sodium gluconate of the
Cl
-free solution was replaced by potassium gluconate with
the remaining components unaltered; for the stromal bath, the sodium
gluconate was replaced by 103 mM NMDG plus 103 mM methanesulfonic acid.
The isoquinolinesulfonamide H-89 was purchased from Calbiochem
(La Jolla, CA) and stored at 5°C in aqueous solution (10 mM). All
other chemicals were from Sigma Chemical (St. Louis, MO) and its
Research Biochemicals affiliate. Solutions of bumetanide (20 mM) and
A-23187 (5 mM) were prepared with ethanol (EtOH) and stored at 5°C.
Also stored at refrigerator temperature as 10 mM stock solutions were
forskolin, IBMX, rolipram, and staurosporine, all in DMSO, as well as
aqueous preparations of MDL-12330A hydrochloride, ouabain octahydrate,
and amphotericin B. Thapsigargin and U-73122 were stored (20°C) as
1 mM solutions in DMSO. All such stocks were used within 3 mo. Maleate
salts of 5-HT and methyl analogs, as well as buspirone HCl,
8-hydroxydipropylaminotetralin HBr (8-OH DPAT), isoproterenol HCl,
propranolol HCl, epinephrine bitartrate, and dibutyryl cAMP (DBcAMP)
were dissolved in water (10 mM) immediately before dilution into the
hemichambers. The serotonergic antagonists WAY-100635 maleate and
4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-fluorobenzamido]ethyl]-piperazine (p-MPPF) dihydrochloride were prepared as aqueous 10 mM solutions, while such concentrations of ketanserin tartrate and spiroxatrine were
made using DMSO. Spiperone HCl was stored at 2 mM in EtOH. All stocks
of the antagonists were consumed within 2-3 days. Acetazolamide and amiloride were prepared as suspensions in amounts equivalent to 10 mM (in EtOH) and 100 mM (in H2O), respectively. The latter readily dissolved on warming (50°C) before dilution. In experiments involving multiple drug treatments, the vehicle concentrations in the
chamber did not exceed 1% for EtOH and 0.1% for DMSO, levels not
found to affect the control electrical parameters.
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RESULTS |
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Previous characterizations of isolated rabbit conjunctival
epithelia in a bicameral Ussing-type arrangement have demonstrated the
coexistence of transport activities that simultaneously mediate Na+ absorption and Cl secretion. The relative
proportions of these oppositely directed functions vary considerably
from one individual preparation to another for reasons that are
unknown, but in general, Cl
transport dominates and
represents on average ~60% of the Isc (33). Given the possibility that 5-HT could serve as a
mediator of either (or both) of these transport processes, initial
experiments determined the effects of the agonist on the control
transepithelial electrical parameters, a topic that had not heretofore
been examined.
Apical additions of 5-HT elicited prompt Isc
reductions and increases in Rt, changes that
occurred in a dose-dependent manner with a calculated EC50
of 7.9 nM (Fig. 1). Because maximal
effects were obtained at 10 µM, this concentration was used in all
subsequent experiments. Additional observations indicated that applying
the agonist to the stromal-side bathing solution resulted in slower and
more variable responses than those obtained apically (not shown).
Presumably, 5-HT does not readily traverse the stroma; it either
reaches receptors on lateral membranes when applied from the apical
direction, and/or its receptors indeed reside at the apical
surface. Virtually identical phenomena were reported earlier in
the case of epinephrine (33). Nevertheless, all data were
acquired from additions of various agents to the apical bathing medium.
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Characterization of the 5-HT effect on transconjunctival
Isc by electrolyte substitutions and transport inhibitors.
The 5-HT-evoked electrical effects most likely resulted from reductions
in conductances across apical Na+ and/or Cl
channels or basolateral K+ channels, given the measured
increase in Rt. In addition,
Isc reductions could also have been due to
inhibitions of conjunctival transporters known to maintain
intracellular K+ and Cl
above equilibrium,
i.e., the Na+-K+ pump, the
Na+-K+-2Cl
cotransporter, and the
coupled activities of the Na+/H+ and
Cl
/HCO
exchangers. To discern the
transport element(s) affected, three distinct experimental approaches
were implemented. One quite simply entailed the bilateral exclusion of
Cl
from the bathing solutions (gluconate substitution).
Under these conditions, the Isc solely reflects
the Na+ absorptive component of the transcellular current,
which can be stimulated by cAMP-elevating maneuvers as a result of an
increase in basolateral K+ conductance(s)
(40). With Cl
absent, introduction of 5-HT
reduced the Isc by 0.5 ± 0.2 (SE) µA/cm2 (an 11% decline from 4.7 ± 0.3 to 4.2 ± 0.5 µA/cm2, n = 5), saliently less
than the inhibitions obtained with complete media
(
Isc =
3.3 ± 0.5 µA/cm2, a 23% change, n = 21). The
limited Isc reduction recorded in the absence of
Cl
was accompanied by a statistically significant
Rt increase of 0.08 ± 0.02 k
· cm2 (a 4% rise from 1.90 ± 0.23 to
1.98 ± 0.26 k
· cm2), suggesting finite
contributions by other conductive pathways to the 5-HT response as
well, with K+ channels the most likely participants.
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Comparative effects of various serotonergic agonists on the
conjunctival transepithelial electrical parameters.
To corroborate the above effects of 5-HT, four additional serotonergic
agonists were arbitrarily selected for determination of their
effectiveness on conjunctival preparations (Table
2). All inhibited the
Isc and increased Rt. The
use of buspirone required a dose 10-fold larger than the others to
attain a maximal response. The effects of the agents used were not
additive, suggesting saturation of native receptors, except in the case
of buspirone, after which additions of 5-HT were found to produce
Isc inhibitions. For illustrative purposes, two
conjunctivae exhibiting marked sensitivity to serotonergics are shown
(Fig. 3). In one case, the inhibitory
response was elicited with 8-OH DPAT, an agonist considered to be
highly selective for the 5-HT1A receptor, a moiety
negatively coupled to adenylyl cyclase activity (2, 11, 15, 28,
48). In the second example, -CH3-5HT, which is
viewed as selective for 5-HT2 receptors positively coupled
to phospholipase C activity (11, 15, 48), was used. However, such noted selectivities should be regarded as tenuous, given
the diversity of 5-HT receptors now known to exist. Consistent with
this, 2-CH3-5HT, considered to be selective for
5-HT3 receptors, which are ligand-gated cation channels
(11, 15, 48), also evoked conspicuous
Isc inhibitions and detectable
Rt increases (Table 2). It was further observed
that the presence of the serotonergic agents did not affect the
succeeding response of the system to the nonselective adrenergic
agonist epinephrine (Fig. 3).
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Absence of an attenuation of the 5-HT-evoked electrical effects by
preexposure to various serotonergic antagonists.
Receptor antagonists were surveyed to determine whether pretreatments
with such agents could diminish the extent of the 5-HT-elicited Isc inhibitions (Table
3). None selected exhibited this
property. Conversely, in the cases of ketanserin and spiperone,
preexposure to these antagonists enhanced the 5-HT effect significantly
as unpaired data. All the antagonists that were tested have reported activities against members of the 5-HT1 receptor family
(6, 15, 48), although ketanserin and spiperone have
markedly lower binding affinities than the others and are generally
regarded as more selective for 5-HT2A receptors (15,
48). An example of the electrical effects of 5-HT on an
epithelium preexposed to spiperone is shown (Fig.
4) in an experiment that also provides indications for the existence of distinct receptor systems, as suggested by the subsequent effects of isoproterenol and propranolol. Although propranolol has been reported to displace labeled 5-HT from
5-HT1A receptors (15, 27), the reversion of
the conjunctival Isc by the -blocker after
isoproterenol stimulation is a repeatable property of this tissue that
occurs independently of the presence of 5-HT (unpublished
observations).
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Direct effects of various serotonergic antagonists on the control
transepithelial electrical parameters.
WAY-100635, regarded as a highly selective antagonist for
5-HT1A receptors (9, 15, 29), did not evoke
significant electrical effects when applied under control conditions
(Table 4). Spiperone elicited mixed
results, with an Isc increase of 1 µA/cm2 obtained in one experiment and decrease of 1.6 µA/cm2 in another; with five additional preparations,
this agent did not generate remarkable Isc
changes (i.e., Isc <0.4
µA/cm2). In contrast, ketanserin and spiroxatrine
produced statistically significant Isc rises.
Conversely, p-MPPF, an agent shown to antagonize 5-HT1A
receptors in some systems (23), elicited clear
Isc decreases in the conjunctiva that were
similar in magnitude to those evoked by 5-HT (Table 4), thereby
suggesting that p-MPPF interacts with conjunctival receptors as if it
were a serotonergic agonist. However, the addition of 5-HT after p-MPPF
led to a further average Isc decrease of 3.4 µA/cm2 in two cases in which this condition was
attempted, a rather typical 5-HT effect that could indicate that these
agents bind to distinct receptors. In two other experiments, ketanserin
was applied after p-MPPF; this maneuver fully reverted the
initial p-MPPF-elicited Isc decrease.
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Ca2+ independence of
5-HT-evoked electrical changes.
The Cl conductance(s) of the conjunctival epithelium can
be increased by elevations of the intracellular concentrations of Ca2+ or cAMP (22, 33, 34). Cellular levels of
the former were augmented in two ways: 1) permeabilizing the
apical surface to the divalent cation with the ionophore A-23187 and
2) inhibiting the Ca2+-ATPase of the
intracellular storage compartments with thapsigargin. Neither treatment
averted the typical effects of 5-HT on the transepithelial electrical
parameters (Fig. 5 illustrates use of the
latter agent). Given that these maneuvers perturb Ca2+
homeostasis irreversibly, the 5-HT-evoked Isc
inhibitions presumably do not result from an increase in extrusion or
sequestration of the cation. Furthermore, it is also unlikely that a
Ca2+ signal downstream from a G protein-linked 5-HT
receptor is involved in the transduction mechanism.
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Differential effects of cAMP-elevating agents on 5-HT-elicited
electrical changes.
Conjunctival epithelia were preconditioned by three experimental
procedures known to increase cellular cAMP concentration: 1)
exposure to the 2-selective agonist isoproterenol,
2) addition of DBcAMP, a cell-permeable form of the
nucleotide, to the apical bath in combination with rolipram, a PDE
inhibitor specific for cAMP-PDE IV (30), and 3)
application of forskolin, a direct stimulator of adenylyl cyclase (Fig.
6). Use of the former condition significantly attenuated (P < 0.05, as unpaired data)
the typical 5-HT-evoked Isc reduction normally
recorded under control conditions (Fig. 6, trace C). In
contrast, 5-HT-elicited electrical responses were not affected when
5-HT was applied to conjunctival currents stimulated by the other two
approaches (Fig. 6, traces A and B). This
phenomenon might be explained if one assumes that forskolin increased
cAMP to levels beyond which a 5-HT-elicited inhibition could be
effective.
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DISCUSSION |
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5-HT, a ubiquitous monoamine with roles in neurotransmission,
paracrine signaling, and inflammatory responses, is widely recognized as a fluid and electrolyte secretagogue in epithelia. Throughout the
gastrointestinal tract, as well as in airway epithelia, 5-HT promotes
secretory activities by stimulating transcellular Cl (or
HCO
) movements directly or by inhibiting absorptive
processes such as neutral transapical Na+ and
Cl
uptake or amiloride-sensitive Na+
conductance (4, 7, 8, 12, 16, 24, 35-37, 49). The
exact nature of the 5-HT receptors, signaling transduction mechanisms,
and affected electrolyte transporters involved in 5-HT-elicited
stimulation of net secretory processes differs depending on the species
and anatomic region characterized. Among ocular tissues, 5-HT is a
known stimulator of electrogenic Cl
transport in the
rabbit corneal epithelium (19). Thus the present results
suggest that the rabbit conjunctiva may be unique among Cl
-secreting and Na+-absorbing epithelia, in
that the indole elicited prompt and sustained inhibitions of
transcellular Cl
movement in the basolateral-to-apical
direction because of the independent downregulation of apical
Cl
and basolateral K+ conductances.
The observed 5-HT-evoked Isc
reductions are likely manifestations of a downregulation in apical
Cl conductance(s), as evidenced by 1) the
marked 5-HT effect under conditions in which the apical bath lacked
Na+, 2) such effect in the presence of
bumetanide under conditions in which transcellular Cl
movement is presumably mediated by the parallel activities of basolaterally located Na+/H+ and
Cl
/HCO
exchangers (41),
3) a similar inhibition of the Cl
-dependent
Isc after complete inhibition of the
Na+/H+ exchanger with amiloride, which in turn
inhibits Cl
/HCO
exchange activity
because of cell acidification, and 4) the extensive
mitigation of 5-HT-elicited Isc reductions with
Cl
-free media as well as with the PKA inhibitor H-89.
Earlier studies on the effects of cAMP elevation (22)
suggested that apical Cl
conductance(s) in the
conjunctiva is gated by PKA. A subset of conjunctival basolateral
K+ conductance(s) is also modulated by PKA
(40), and the present study obtained evidence for an
effect by 5-HT on K+ channels as well.
The inhibitions of the Cl-dependent
Isc could have solely arisen from a 5-HT-induced
reduction in the driving force for apical Cl
secretion
secondary to K+ channel closure. However, the fact that an
effect by 5-HT after K+ channel blockade was not observed
in the absence of Cl
but was indeed obtained with the
anion present (Table 1) further supports the likelihood that 5-HT also
directly regulates apical Cl
channels in addition to
basolateral K+ channels.
Given the Ca2+ independence of the 5-HT-elicited electrical
effects and the fact that Ca2+ and cAMP are the
intracellular messengers most commonly found to be involved in the
gating of Cl and K+ channels, the present
results are most plausibly explained as a consequence of a 5-HT-evoked
reduction in cAMP concentration. If this is so, the conjunctiva
expresses 5-HT receptors of the type 1 family, which are negatively
linked to adenylyl cyclase activity.
Seven families of 5-HT receptors are recognized, with 15 subtypes
identified (11). Furthermore, such subtypes can in some cases exist in several isoforms due to RNA editing or alternative splicing (18). One consequence of the extensive diversity
of 5-HT receptors is that the degree of selectivity of ligands regarded as specific for a particular subtype appears to become questionable due
to their newly realized affinities for recently discovered receptors.
As such, a pharmacological characterization of the putative 5-HT
receptors present in the conjunctiva based on functional criteria
(i.e., agonist and antagonist rank order in affecting the electrical
parameters) would not be efficient and was not attempted. Further
studies in this regard using radioligand binding assays on membrane
fractions to determine apparent affinity values of various ligands
would be more appropriate (6). Measurements of second
messengers, e.g., cAMP, and/or G protein activation assays (29,
38) may be similarly useful but are beyond the scope of this
electrophysiological study.
The ligands that were screened in the present study were arbitrarily chosen from among several possibilities, but with the presumption in mind that the conjunctiva might harbor 5-HT1A receptors. Such receptors had been identified in the ocular ciliary epithelium (2) and in cultured retinal pigmented epithelial cells (28). It was posited a priori, for example, that WAY-100635, given its high selectivity for this subtype (9), might attenuate the 5-HT-elicited current reduction and that ketanserin would be ineffective. However, none of the antagonists selected affected the evoked 5-HT effect, and all the agonists tested inhibited the Isc and increased Rt.
It is possible that the lack of a mitigation in the 5-HT effect subsequent to antagonist preexposure may have been due to the use of a supramaximal dose of 5-HT. However, pretreatment with an antagonist at the same dose as sequentially applied 5-HT should have resulted in a measurable attenuation in the 5-HT response if both agents were acting on the same site. Either 5-HT completely displaced the antagonist, or discrete receptors exist. Nevertheless, applying 5-HT at lower doses would result in smaller current changes and greater uncertainty (witness the magnitude of the standard errors obtained).
The Isc stimulations produced by the antagonists ketanserin, spiroxatrine, and, in one preparation, spiperone, suggest that these agents may be operating as "inverse" agonists (29). This phenomenon has been attributed to an inhibition of "constitutive" coupling between a receptor and a G protein. Alternatively, but perhaps less likely, the antagonists displaced endogenous 5-HT present at the receptors.
The notion that the conjunctival epithelium might express
5-HT1 receptors rests on the assumption that reductions in
cAMP levels are the most cogent manner in which K+ and
Cl conductances could be downregulated. Hypothetically,
one could also posit that phosphatase activity could have been
augmented. Nevertheless, the present data uncovered the paradox that
although 5-HT-evoked Isc reductions were not
affected by pretreatments with isoproterenol or the combination of
DBcAMP and rolipram, preexposure to forskolin significantly attenuated
the 5-HT effect. These results illustrate complexities commonly
encountered in functional measurements and are reminiscent of a
phenomenon described with cultured human retinal pigmented epithelial
cells (28), in which, conversely, 5-HT reduced cAMP levels
stimulated by forskolin but not by isoproterenol or adenosine.
In regard to the present results with the conjunctiva, forskolin may have increased cAMP levels far beyond what is needed to fully stimulate epithelial conductances. Any putative reduction in cAMP concentration by a 5-HT-triggered mechanism could bring levels down to values still above a saturation level. This may not be the case for the other cAMP-elevating agents used in this study.
More problematic is the fact that a physiological role for the apparent
5-HT receptors that were discerned by this study is not clearly
evident. Nor is the location of the disclosed receptor(s), i.e., apical
vs. basolateral aspects of the epithelium, definitively stated from the
experiments presented because of the limitations of the preparation. An
apical presence would imply that such receptors are activated by
changes in 5-HT levels in tears (25). Conversely, a
basolateral location might suggest that 5-HT, possibly released by
stromal mast cells, could serve as an inflammatory mediator under
conditions such as allergic conjunctivitis and influence the secretory
activity of the epithelium. This possibility is not inherently
intuitive, given that mast cells also liberate histamine and
prostaglandins, agents that might be expected to stimulate
Cl transport.
The central role of conjunctival goblet cells is to produce the mucus
layer of the tear film, which protects the corneal and conjunctival
epithelia from various environmental insults. However, in this case as
well, there does not necessarily appear to be a connection between
5-HT-elicited goblet cell mucin secretion (17) and
transepithelial Cl transport. ACh also stimulates mucin
secretion (31) without having an effect on the
transepithelial electrical parameters (unpublished observations).
Briefly, a novel inhibitory effect by 5-HT on the Cl
secretory activity of the rabbit conjunctival epithelium was evinced, apparently rendering to this tissue a unique status among
Cl
-secreting epithelia. Further work is needed to
characterize this unusual property, given the prospect that this tissue
may provide an opportunity to study, via biochemical and molecular
approaches, a previously unobserved effect of 5-HT.
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ACKNOWLEDGEMENTS |
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This work was supported by National Eye Institute Grants EY-11631 and EY-01867 and by an unrestricted grant from Research to Prevent Blindness (New York, NY).
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. J. Alvarez, Dept. of Ophthalmology, Mount Sinai School of Medicine, 100th St. and 5th Ave., New York, NY 10029 (E-mail: larry.alvarez{at}mssm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 June 2000; accepted in final form 25 September 2000.
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