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INTRODUCTION |
Prostaglandin E2 receptors (EP
receptors)1 have been
classified into four general subtypes, EP1, EP2, EP3, and EP4, based on cloning and pharmacological manipulations (1, 2). These receptors are
G-protein-coupled, and binding of agonists results in activation
of various transduction cascades depending on the receptor subtype
activated and the cells being studied. Activation of the EP1 receptor
in kidney tubule cells increases the concentration of intracellular
calcium, phosphoinositol turnover, and PKC activity (3, 4). EP2 and EP4
receptors are coupled through Gs (1) to increase
intracellular cAMP in a number of preparations (5-8). The EP3 receptor
undergoes post-transcriptional RNA splicing to produce multiple EP3
isoforms, and activation of these splice variants can increase calcium
mobilization or either stimulate or inhibit cAMP production (9-11).
Because subtypes of the EP receptor can be linked to different
transduction cascades in different types of cells, it is critical to
determine which receptors and receptor-associated signal transduction
pathways are responsible for specific physiological actions of E-series prostaglandins.
Although PGE2 has a number of diverse physiological actions
(12), its role in pain and inflammation is of primary importance. Indeed, both the analgesic and the anti-inflammatory actions of the
nonsteroidal anti-inflammatory drugs are attributed to their ability to inhibit prostaglandin synthesis (13). In addition, PGE2 is produced at sites of tissue injury (14), and
administration of prostaglandins produces vasodilation and edema and
augments pain perception in animals and in humans (15). The
proinflammatory actions of PGE2 result, in part, from a
direct action of this eicosanoid on sensory neurons. In in
situ preparations, E-series prostaglandins augment the firing of
sensory neurons in response to noxious stimuli (16, 17). Furthermore,
exposing sensory neurons in culture to PGE2 increases cAMP
content (18), increases the number of action potentials elicited by a
depolarizing stimulus (19), and augments the evoked release of the
neuropeptides (18, 20).
Despite the fact that PGE2-induced sensitization of sensory
neurons has been extensively studied, the EP receptors expressed on
sensory neurons and which subtypes mediate sensitization remain unknown. Consequently, we used RT-PCR to ascertain which receptor subtype mRNAs are expressed in sensory neurons. We also used
antisense oligonucleotides to selectively reduce expression of EP
receptor subtypes and examine whether this "knockdown" alters
PGE2-induced increases in cAMP and augmentation of release
of immunoreactive substance P (iSP) and immunoreactive calcitonin
gene-related peptide (iCGRP). We choose to use antisense, because EP
receptor subtypes have highly conserved structures with ligands often
activating multiple receptor subtypes (1, 2), and thus there are
limited pharmacological tools to distinguish subtypes. Our data provide novel evidence that activation of the EP3C and EP4 receptors mediates the PGE2-induced sensitization of sensory neurons,
suggesting that multiple receptor subtypes can subserve the same function.
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EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium,
L-glutamine, penicillin/streptomycin, Hanks' balanced salt
solution, and fetal bovine serum were obtained from Life Technologies,
Inc. Nerve growth factor was purchased from Harlan Bioproducts for
Science, Inc. (Indianapolis, IN). Prostaglandin E2,
butaprost, 1-OH-PGE1, and sulprostone were obtained from
Cayman Chemical Co. (Ann Arbor, MI). Peptides were obtained from
Peninsula Laboratory (Belmont, CA). Capsaicin, forskolin,
3-isobutyl-1-methylxanthine (IBMX), and other routine chemicals were
purchased from Sigma. Capsaicin and prostaglandins were initially
dissolved in 1-methyl, 2-pyrrolidione (Sigma) to a concentration of 10 mM and then diluted to appropriate concentration in
perfusion buffer. In no instances does this vehicle at the dilutions
used alter cAMP or neuropeptide release. Substance P antiserum was
raised in our laboratory (21), whereas the CGRP antiserum; the EP1,
EP3A, and EP4 antiserum; the EP2 antiserum; the IP antiserum;
and the EP3 antiserum, which recognizes a conserved region of EP3
receptors, were generous gifts from Michael J. Iadarola (National
Institutes of Health, Bethesda, MD), Hitoshi Shichi (Wayne State
University), John Regan (University of Arizona), Micah Doty (Cayman
Chemicals), and Hsin-Hsiung Tai (University of Kentucky), respectively.
Rat kidney cDNA was a gift from H. R. Besch, Jr. (Indiana
University). Actin antibody was purchased from Chemicon (Temecula, CA).
Isolation and Culture of Rat Sensory Neurons--
Cultures of
embryonic rat sensory neurons were prepared using a modification of
previously described methods (20). Briefly, sensory neurons were
dissociated from the dorsal root ganglion of 15-17-day rat embryos
using trypsin and mechanical agitation. Sensory neurons were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 2 mM glutamine, 250 ng/ml nerve growth
factor, 50 µg/ml penicillin and streptomycin, and the mitotic
inhibitors, 5-fluoro-2-deoxyuridine (50 µM) and uridine
(150 µM). Cells were plated in 24-well
poly-D-lysine (100 µg/ml)-coated culture plates at
~150,000 cells/well. The cells were maintained at 37 °C in a 5%
CO2 atmosphere for 9-11 days, and growth medium was
changed every second day.
Cultures of adult dorsal root ganglia cells were prepared using a
modification of the method of Lindsay (22). Male Harlan Sprague-Dawley
rats weighing 150-250 g were sacrificed with CO2. The
dorsal root ganglias from each rat were collected in sterile calcium-
and magnesium-free modified Hanks' balanced salt solution. The dorsal
root ganglias were digested with 0.125% collagenase in growth
medium consisting of F-12 supplemented with 10% horse serum, 2 mM glutamine, 50 µg/ml penicillin and streptomycin, and the mitotic inhibitors 5-fluoro-2-deoxyuridine (50 µM)
and uridine (150 µM) at 37 °C for 2 h. Cells were
dissociated by mechanical agitation and plated in 24-well culture
plates coated with poly-D-lysine and laminin at ~15,000
cells/well. The cultures were maintained at 37 °C in an atmosphere
of 3% CO2 for 7 days in the presence of 250 ng/ml nerve
growth factor. The Animal Care and Use Committee (Indiana University
School of Medicine, Indianapolis, IN) approved all procedures used in
these studies.
Peptide Release and cAMP Production in Sensory Neurons in
Culture--
For the release experiments, cells were washed twice with
HEPES buffer containing 25 mM HEPES, 135 mM
NaCl, 3.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 3.3 mM
D-glucose, 0.1 mM ascorbic acid, 1 µM phosphoramidon, and 0.1% bovine serum albumin, pH
7.4. The cells then were exposed to HEPES buffer or HEPES plus 100 nM PGE2 or 1 µM forskolin for 10 min prior to and throughout the first and second incubations
(i.e. basal and stimulated). The doses and times of exposure
for PGE2 and forskolin were based on our previous work with
prostanoids (18, 20). Peptide release was evoked during the second
10-min incubation by exposing cultures to 30 nM capsaicin.
Following the 10-min stimulation period, basal release was
reestablished by exposing the cells to HEPES buffer for 10 min. The
supernatants were collected following each incubation, and iSP and
iCGRP were quantitated using radioimmunoassay as previously described
(20).
For cAMP experiments, cells were washed twice with 0.5 ml of HEPES
buffer containing the phosphodiesterase inhibitor, IBMX. Following a
20-min incubation in 0.5 ml of HEPES buffer containing 2 mM
IBMX in the presence or absence of forskolin, PGE2, or
other prostanoid agonists, the cells were collected in 0.5 ml of 0.1 M HCl, boiled for 3 min, and pelleted. The supernatant was
decanted, frozen, and lyophilized. The content of the immunoreactive
cAMP (icAMP) was assayed by radioimmunoassay (PerkinElmer Life
Sciences) using the nonacetylated protocol. Analysis of variance was
used to compare the effects of different treatments on the neuropeptide release and cAMP production, and if a significant difference was observed, the Student-Newman-Keuls post hoc test
was performed. The significance level for all tests was set at
p < 0.05.
Immunoblotting--
Cells were washed twice in sterile
phosphate-buffered saline and disrupted by freezing in 100-200 µl of
lysis buffer containing 125 mM Tris-HCl, pH 6.8, 4% SDS,
20% glycerol, 200 mM dithiothreitol, 0.02% bromphenol
blue. Equivalent amounts of protein from cell lysates, as determined by
using the Bio-Rad protein detection kit, were separated on 12%
SDS-polyacrylamide gels and electrophoretically transferred to
nitrocellulose. The membrane was blocked with 5% milk in Tris-buffered
saline with 0.2% Tween 20 overnight and incubated for 24 h at
4 °C with polyclonal antibody to the EP1, EP2, EP3A, EP3, EP4, and
IP receptor subtypes or to actin followed by incubation with the
appropriate horseradish peroxidase-conjugated secondary antibody for
1 h. Immunoreactive bands were developed by an ECL kit and
visualized by exposure to Eastman Kodak Co. LS X-Omat film.
Quantification of the immunoreactive bands was analyzed using NIH-Image
software. Student's t test was used to compare effects of
antisense or missense treatment on receptor expression.
RNA Isolation and Reverse Transcription-PCR--
Total RNA was
isolated from sensory neurons in culture using a QuickPrep Total RNA
Extraction Kit (Amersham Pharmacia Biotech). Messenger RNA was
reverse-transcribed into cDNA using Superscript II reverse
transcriptase (Superscript II RNase H Reverse Transcriptase Kit; Life
Technologies). Total RNA (~1-5 µg) and 0.5 µg of
oligo(dT)12-18 primer were heated to 70 °C for 10 min
and briefly chilled on ice. After primer annealing, the following were
added: 50 mM Tris-HCl, pH 8.8, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTP, and 40 units of RNasin ribonuclease inhibitor. The
reaction incubated for 50 min at 42 °C and then for 15 min at
70 °C. An aliquot of each reaction was subsequently used as template
for a PCR.
Primer Preparation and PCR--
The primers were designed
to be selective for the PGE2 and PGI2
receptors, and the sequences used were as follows: EP1 (336 base pairs
(bp)), 5'-CGCAGGGTTCACGCACACGA-3' (nucleotides 865-884) and
5'-CACTGTGCCGGGAACTACGC-3' (nucleotides 1182-1201) (23); EP2 (369 bp),
5'-CCGCGCGTGTACCTATTTCGC-3' (nucleotides 363-383) and
5'-GCTCCGAAGCTGCATGCGAA-3' (nucleotides 713-732)
(GenBankTM accession number U94708); EP3A (309 bp),
5'-GCTGTCTGTGCTCGCCTT-3' (nucleotides 471-490) and
5'-CCATAAGCTGGATAG-3' (nucleotides 767-784) (24); EP3B (453 bp),
5'-CTGTGCTCGCCTTCGCGC-3' (nucleotides 567-586) and
5'-GCCAGGCGAACGGCGATT-3' (nucleotides 1003-1020)
(GenBankTM accession number D29969); EP3C (413 bp),
5'-TGGTGGTGACCTTTGCCTGCAACCTGGC-3' (nucleotides 653-680) and
5'-CAAGGAGATGGCCTGCCCTTTCTGTTGG-3' (nucleotides 1038-1065) (25);
EP4 (423 bp), 5'-TTCCGCTCGTGGTGCGAGTGTTC-3' (nucleotides
941-963) and 5'-GAGGTGGTGTCTGCTTGGGTCAG-3' (nucleotides 1342-1364) (23); IP (431 bp), 5'-GCATCCTGGGTGCCCGACG-3'
(nucleotides 276-295) and 5'-CAGGCTGGGGGGAACGCAT-3' (nucleotides
688-707) (GenBankTM accession number D28966); and rat
histone H3.3 (231 bp), 5'-GCAAGAGTGCGCCCTCTACTG-3' (nucleotides
80-100) and 5'-GGCCTCACTTGCCTCCTGCAA-3' (nucleotides 274-294) (26).
The PCR primers for the EP3C receptor are based on primers for an EP
receptor originally classified as EP3B (25); however, the receptor is
identical to the mouse EP3C receptor (11). The PCR mixture contained a
cDNA template derived from total RNA, 1 unit of recombinant
Taq DNA polymerase (Life Technologies, Inc.), 50 pmol each
of 5'- and 3'-primers (Life Technologies), 0.2 mM dNTP, in
a buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgSO4 in a volume of
50 µl. The PCR was performed using a PerkinElmer Life Sciences
Thermocycler (model 2400) as follows: 94 °C for 120 s and then
37 cycles of 94 °C for 45 s, 60 °C for 45 s, and
72 °C for 120 s followed by 74 °C for 10 min. Samples were
applied on 1% agarose gel prestained with 0.5 µg/ml ethidium bromide.
Phosphorothioate Oligonucleotides--
Antisense
oligonucleotides were designed to be complementary to the
PGE2 receptor subtypes EP2, EP3C, and EP4. Missense control oligonucleotides were also synthesized to correspond to antisense sequences except for pairs of bases that have been switched.
Oligonucleotide sequences were as follows: EP2, antisense
5'-GCCTGGAGTCATTGA-3' (bases 56-70) and missense
5'-CGCGTGAGTCTATGA-3'; EP3C, antisense 5'-GATGGCCTGCCCTTT-3'
(bases 1101-1125) and missense 5'-TAGGGCGCTCCTCCTT-3'; EP4, antisense
5-GACTCCGGGGATGGA-3' (bases 4-18) and missense 5'-GACCTCGGGAGTGAG-3'.
Oligonucleotides were synthesized (Life Technologies Custom Primers)
with phosphorothioate linkages to prevent nuclease degradation.
Sensory neurons in culture were exposed to 1 µM antisense
or missense oligonucleotide in growth medium or growth medium
alone for 48 h with a change of medium and oligonucleotide at
24 h.
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RESULTS |
Identification of PGE2 Receptor Subtypes in Sensory
Neurons--
To ascertain which PGE2 receptor subtype
mRNAs were found in sensory neurons, total RNA from cultured
embryonic or adult sensory neurons was reverse-transcribed and
subjected to PCR in the presence of primers for the PGE2
receptor subtypes EP1, EP2, EP3A, EP3B, EP3C, EP4, the PGI2
receptor, IP, or histone protein H3.3. Fig. 1, A and B, depicts
the PCR products from embryonic and adult neurons, corresponding to the
EP1, EP2, EP3C, EP4, and IP receptors that were detected in the gel at
the expected sizes of 336, 369, 413, 423, and 431 bp, respectively. PCR
products for the EP3A and EP3B receptor from sensory neurons were not
detected. To confirm that this lack of PCR products to the EP3A and
EP3B receptor subtypes was not secondary to amplification problems, we
performed RT-PCR under identical conditions using total RNA isolated
from the rat kidney, since EP3A and EP3B receptors are highly expressed
in this tissue (24, 27). As can be seen in Fig. 1C, EP3A and EP3B receptor PCR products were detected with approximate sizes of 309 and 453 bp, respectively. As the external control, replicate samples of histone H3.3 PCR product were detected in sensory neurons with an expected size of 231 bp.

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Fig. 1.
Detection of PGE2 receptor
subtype mRNA and protein in isolated sensory neurons. Total
RNA from embryonic (A) or adult (B) sensory
neurons was isolated and subjected to RT-PCR, and PCR products for the
EP1 (1), EP2 (2), EP3C (3C), EP4
(4), and IP (I) receptor subtypes were detected,
as indicated. DNA size markers are shown in the lanes marked
S. Histone H3.3 (H) was simultaneously amplified
with PCR as an external control. C shows detection of
PGE2 receptor EP3A (3A) and EP3B (3B)
subtype mRNA in kidney (K) but not sensory neurons
(SN) by RT-PCR. RNA was extracted from kidney (K)
or embryonic sensory neurons (SN). D represents
an immunoblot of PGE2 receptor subtypes EP1, EP2, EP3, and
EP4 and the IP2 receptor from protein isolated from
embryonic sensory neurons.
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The expression of PGE2 receptor proteins in sensory neurons
also were examined using antibodies that recognize the EP1, EP2, EP3,
EP4, and IP receptors. Immunoblots with molecular masses of ~65, 67, 60, 63, and 66 kDa were detected when protein isolated from embryonic
sensory neurons was separated by gel electrophoresis (Fig.
1D). These molecular masses correspond to the sizes
of the EP1, EP2, EP3, EP4, and IP receptors (28-30, 58, 59). No
immunoreactive band for the EP3A receptor was expressed by sensory
neurons (data not shown), further supporting the RT-PCR finding that
the EP3A receptor was not expressed by sensory neurons.
Identification of PGE2 Receptor Subtypes Involved in
the PGE2-stimulated cAMP Production--
We used antisense
oligonucleotides that inhibit formation of specific EP receptor
subtypes to determine which receptors mediate PGE2-induced
increases in the production of cAMP. Sensory neurons in culture were
exposed for 48 h to medium alone or to medium containing 1 µM antisense or missense oligonucleotide to
the EP2, EP3C, and/or EP4, and then the PGE2-stimulated
production of cAMP was measured. We focused on these receptor subtypes
because cloned rat or mouse EP2, EP3C, and EP4 receptors have been
shown to increase cAMP production (5, 9-11). In untreated neuronal
cultures, exposure to 100 nM PGE2 and 2 mM IBMX for 20 min significantly increased the content of
icAMP 2.5-fold over cells treated only with IBMX (Fig.
2, A and B). In
cultures treated with individual antisense oligonucleotides directed
toward mRNA for EP2, EP3C, or EP4 receptor subtypes or to missense
oligonucleotides, 100 nM PGE2 also
significantly increased cAMP production (Fig. 2A), suggesting that reducing expression of individual receptor subtypes had
no effect on the PGE2-stimulated production of cAMP.
Consequently, we further examined whether simultaneously decreasing
expression of multiple receptor subtypes would attenuate the actions of
PGE2. When neuronal cultures were pretreated for 48 h
with 1 µM of each antisense directed at mRNA for the
EP2, EP3C, and EP4 receptors, the effects of 100 nM
PGE2 were completely abolished in that icAMP levels were
3.1 ± 0.5 pmol/well (n = 17; Fig. 2B).
In a similar manner, when sensory neurons were exposed to antisense
oligonucleotides directed toward mRNA for only the EP3C and EP4
receptor subtypes, exposure to 100 nM PGE2 did
not increase the content of icAMP (3.1 ± 0.5 pmol/well,
n = 13). In contrast, antisense oligonucleotides directed toward the EP2 and EP4 receptor subtypes did not significantly attenuate the effect of the prostanoid, since in these cultures icAMP
was elevated to 5.8 ± 0.5 pmol/well (n = 9).
Exposing sensory neurons to any combination of missense
oligonucleotides had no effect on the PGE2-stimulated
production of icAMP. These results suggest that simultaneous loss of
the EP3C and EP4 receptor subtypes is necessary to prevent
PGE2 from increasing the formation of cAMP in sensory
neurons.

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Fig. 2.
Effects of antisense oligonucleotides
treatment on PGE2-induced increases in icAMP in sensory
neurons. Columns represent the mean ± S.E. of
icAMP (iCyclic AMP) content in pmol/well for the indicated
number of wells. The open columns represent cells
incubated in HEPES buffer containing 2 mM IBMX for 20 min
(basal), whereas the hatched columns represent
cells treated with 100 nM PGE2 and 2 mM IBMX for 20 min. Shaded columns
represent icAMP from cultures preexposed to either individual antisense
or missense oligonucleotides (A) or combinations of
oligonucleotides (B), as indicated. An asterisk
represents a significant difference between basal and
PGE2-stimulated content of icAMP using analysis of variance
with Neuman-Keuls as the post hoc test
(p < 0.05).
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Because it is possible that the inhibitory effect of the combination of
antisense oligonucleotides could reflect a lack of specificity or
toxicity (31), we examined whether cultures exposed to EP3C and EP4
antisense oligonucleotides were capable of producing cAMP by treating
cells with forskolin, a direct activator of adenylyl cyclase. In
untreated neuronal cultures, a 20-min exposure to 1 µM
forskolin in the presence of 2 mM IBMX increased the
content of icAMP from 2.2 ± 0.3 pmol/well (n = 20) to 30.2 ± 1.9 pmol/well (n = 20; Fig.
3). In neuronal cultures pretreated for
48 h with antisense oligonucleotides to the EP3C and EP4 subtype
mRNA or missenses, 1 µM forskolin increased the
content of icAMP to 29.3 ± 3.3 pmol/well (n = 17)
and to 35.1 ± 3.8 pmol/well (n = 12), respectively. Thus, treating cells with antisense or missense oligonucleotides did not block the ability of the sensory neurons to
produce cAMP.

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Fig. 3.
Effects of antisense oligonucleotide
treatment on forskolin-induced increases in icAMP in sensory
neurons. Columns represent the mean ± S.E. of
icAMP (iCyclic AMP) content in pmol/well for the indicated
number of wells. The open columns represent cells
incubated in HEPES buffer containing 2 mM IBMX for 20 min
(basal), whereas the hatched columns represent
cells treated with 1 µM forskolin and 2 mM IBMX for 20 min. Shaded columns
represent icAMP from cultures preexposed to either antisense or
missense to EP3C and EP4 receptor subtypes mRNA as indicated.
Asterisks represent a significant difference between basal
and forskolin-stimulated levels of icAMP using analysis of variance
with Neuman-Keuls as the post hoc test
(p < 0.05).
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Since our antisense experiments suggested that activation of either the
EP3C or EP4 receptor results in the enhanced production of cAMP, we
addressed whether the potential activation of these receptors with the
putative EP receptor agonists, butaprost, 1-OH-PGE1, or
sulprostone, would similarly enhance cAMP production in sensory neurons. As in the above experiments, treating cells with 100 nM PGE2 significantly increased icAMP content
1.4-fold from 4.0 ± 0.2 pmol/well (n = 8) to
5.9 ± 0.6 (n = 8). In contrast, exposure to a 100 nM concentration of the putative EP1/EP3 agonist,
sulprostone, or either a 100 nM or 1 µM
concentration of the EP2/EP4 agonist, 1-OH-PGE1, had no
effect on basal icAMP production; icAMP content was 2.7 ± 0.5 pmol/well (n = 8), 3.4 ± 0.2 pmol/well
(n = 8), or 4.2 ± 0.4 pmol/well
(n = 8), respectively. Exposure to a 100 nM
concentration of the EP2 agonist, butaprost, did not increase icAMP
production (4.8 ± 0.4 pmol/well, n = 8), whereas
exposure to 1 µM butaprost significantly enhanced icAMP
levels to 10.6 ± 1.0 pmol/well (n = 8).
Since micromolar concentrations of butaprost can bind to prostaglandin
receptor subtypes other than EP2 (57), we addressed whether the
increase in cAMP content stimulated by 1 µM butaprost was
secondary to actions at the EP2, EP3C, or EP4 receptor subtype. Pretreating neuronal cultures for 48 h with 1 µM
antisense directed at the EP2 receptor or at the EP3C and EP4
receptors in combination significantly reduced the butaprost-induced
increase in icAMP content. Exposure to antisense directed toward the
EP2 receptor decreased the 1 µM butaprost-stimulated
icAMP content from 9.2 ± 0.8 pmol/well (n = 11)
in untreated cells to 5.2 ± 0.3 pmol/well (n = 8), and exposure to antisense toward the EP3C and EP4 receptors also
reduced the icAMP content to 6.4 ± 1.1 pmol/well
(n = 9). Although the butaprost-induced increase in
cAMP content was reduced by either treatment, the agonist still
significantly increased the levels of the second messenger above
control. Exposing sensory neurons to missense oligonucleotides had no
effect on the butaprost-stimulated icAMP.
Antisense Oligonucleotides Diminish the Expression of
PGE2 Receptor Subtypes EP2, EP3, and EP4--
To
substantiate that exposure to antisense oligonucleotides attenuated
expression of EP2, EP3, and EP4 receptors, protein extracts from
untreated cells or cells treated with antisense or missense were
immunoblotted with antisera raised against specific EP receptor
epitopes. Exposing sensory neurons to increasing concentrations of
antisense for 48 h resulted in a
concentration-dependent decrease in EP receptor expression
with a maximal inhibition at 1 µM antisense (data not
shown). After 48 h of treatment with 1 µM antisense, the optical density of immunoreactive EP2 bands in cells exposed to EP2
antisense was decreased by 71 ± 7% (n = 2),
immunoreactive EP3 bands were decreased by 85 ± 9%
(n = 2) in EP3C antisense-treated cells, and
immunoreactive EP4 expression was decreased 69 ± 8% (n = 2) in cells treated with antisense to EP4 (Fig.
4A). In sensory neurons
exposed to 1 µM antisense to the EP3C and EP4 receptor for 48 h, conditions that abolished PGE2-induced icAMP
production, the immunoreactivity of EP3 and EP4 receptor protein was
significantly reduced compared with protein from untreated cultures of
sensory neurons, whereas immunoreactivity of EP2 and IP receptors was not affected (Fig. 4B). Furthermore, exposure to antisense
to the EP2 receptor did not affect EP3C and EP4 receptor expression (data not shown). Immunoreactive EP3 bands were decreased by 83 ± 8% (n = 5), and immunoreactive EP4 expression was
decreased 76 ± 6% (n = 5) in cells treated with
antisense to EP3C and EP4 receptor simultaneously (Fig. 4B).
In contrast, there is no significant reduction in receptor expression
in neurons treated with missense oligonucleotides (Fig. 4B).
To confirm that equal amounts of protein were loaded onto the gel, the
expression of actin also was examined. As illustrated in Fig. 4,
A and B, the relative expression of actin did not
appear to be affected by exposure to antisense or missense
oligonucleotides.

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Fig. 4.
Expression of PGE2 receptor
subtypes EP2, EP3, and EP4 by sensory neurons after antisense
treatment. A, exposure to 1.0 µM
antisense oligonucleotide to EP2, EP3C, or EP4 receptors reduces
receptor expression. B, neuronal cultures were exposed to
medium alone (Con) or medium supplemented with 1 µM missense (MS) or antisense (AS)
oligonucleotide to EP3C and EP4 receptors simultaneously. Total protein
was isolated from sensory neurons grown in culture. 50 µg of the
total protein that was extracted from the cultures was separated using
a 12% acrylamide gel and transferred to nitrocellulose. After exposure
to primary antibodies, immunoreactive bands for the EP2, the EP3, the
EP4, and the IP receptor or for actin were detected.
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Identification of PGE2 Receptor Subtypes Mediating the
PGE2-induced Augmentation of Peptide Release--
Because
EP3C and EP4 receptor subtypes are required for the
PGE2-stimulated production of icAMP, we sought to determine
whether these receptor subtypes also mediated PGE2-induced
facilitation of the evoked release of neuropeptides. After pretreatment
with medium alone or medium containing 1 µM of
antisense or missense oligonucleotide directed toward the EP3C and EP4
receptor subtypes for 48 h, sensory neurons were exposed for 10 min to either buffer alone or buffer containing 100 nM
PGE2, followed by a 10-min exposure to buffer containing 30 nM capsaicin in the presence or absence of
PGE2. As can be seen in Fig.
5, exposing neurons to capsaicin significantly increased the release of iSP from 3.5 ± 0.2 to
22.1 ± 1.1 fmol/well/10 min (n = 18) and
increased iCGRP release from 33.3 ± 3.1 to 211.5 ± 13.2 fmol/well/10 min (n = 18). In cultures not exposed to
antisense oligonucleotides, treatment with 100 nM
PGE2 did not directly stimulate peptide release but
significantly augmented the capsaicin-stimulated release of both
peptides by 1.4-fold (Fig. 5). When sensory neurons were preexposed to
antisense oligonucleotides directed toward the EP3C and EP4 receptor
subtype mRNA, the PGE2-augmented release of
neuropeptide was abolished (Fig. 5). In these neurons, capsaicin-evoked
release in the presence of PGE2 was 22.8 ± 1.4 fmol/well/10 min (n = 18) for iSP and 226.0 ± 27.1 fmol/well/10 min (n = 17) for iCGRP. Pretreating
cells with missense oligonucleotides did not effect the ability of
PGE2 to augment peptide release (Fig. 5). In addition,
neither antisense nor missense treatment had any significant effect on
capsaicin-stimulated release in the absence of PGE2 (Table
I).

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Fig. 5.
Treatment with antisense oligonucleotide to
EP3C and EP4 subtypes abolishes the PGE2-augmented peptide
release. As indicated, cells were exposed to medium alone
or medium supplemented with 1 µM antisense or missense
oligonucleotides to EP3C and EP4 receptors, simultaneously. The
ordinate represents the mean ± S.E. of immunoreactive
substance P (iSubstance P; top panel)
or of immunoreactive CGRP (bottom panel) released
in fmol/well/10 min of incubation. Open columns
show release of neuropeptide when cells were exposed to HEPES buffer
alone, and hatched columns show release when
cells were exposed to HEPES buffer in the presence of 100 nM PGE2. Shaded columns
show release of neuropeptide from cells stimulated with 30 nM capsaicin (CAP) in the absence or presence of
PGE2 as indicated. An asterisk indicates a
significant difference from basal release, and a cross
indicates a significant difference from capsaicin-stimulated release
using an analysis of variance with Neuman-Keuls as post
hoc test (p < 0.05).
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Table I
Effects of individual antisense oligonucleotides on
PGE2-induced augmentation of iSP and iCGRP release from rat
sensory neurons
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In an additional series of experiments, we examined whether the
PGE2-augmented release could be blocked by inhibiting
expression of either the EP3C or EP4 receptors independently. As can be
seen in Table I, exposure to PGE2 in untreated cells did
not alter basal release but significantly augmented the
capsaicin-stimulated release of iSP and iCGRP by 1.5-fold. Unlike the
simultaneous antisense exposure toward the EP3C and EP4 subtypes (Fig.
5), antisense treatment toward either the EP3C or the EP4 receptor subtype individually did not effect the ability of PGE2 to
augment the capsaicin-evoked release of iSP and iCGRP (Table I).
Activating the production of cAMP with forskolin also augments evoked
release of neuropeptides (18). Consequently, as a way of confirming
that antisense inhibition of prostaglandin sensitization was not
secondary to toxicity, we examined whether this action of forskolin was
inhibited by antisense treatment. In untreated cells, exposure to 1 µM forskolin for 10 min prior to and throughout stimulation with capsaicin did not alter basal peptide release but
significantly augmented the capsaicin-stimulated release of iSP and
iCGRP by 1.6- and 1.7-fold, respectively (Fig.
6). In sensory neurons preexposed to
antisense oligonucleotides directed toward the EP3C and EP4 receptor
subtypes or to missense oligonucleotides, forskolin still significantly
augmented the capsaicin-evoked release (Fig. 6). Thus, antisense
treatment inhibits the augmented release of neuropeptides induced by
PGE2 but does not affect the ability of forskolin to
sensitize sensory neurons.

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Fig. 6.
Treatment with antisense oligonucleotides to
EP3C and EP4 subtypes does not affect the forskolin-augmented peptide
release. As indicated, neurons were exposed to medium alone
or medium supplemented with 1 µM antisense or missense
oligonucleotides to EP3C and EP4 receptors simultaneously. The
ordinate represents the mean ± S.E. of immunoreactive
substance P (iSubstance P; top panel)
or of immunoreactive CGRP (bottom panel) released
in fmol/well/10 min of incubation. Open columns
show release of neuropeptide when cells were exposed to HEPES buffer
alone, and hatched columns show release when
cells were exposed to HEPES buffer in the presence of 1 µM forskolin. Shaded columns show
release of neuropeptide from cells stimulated with 30 nM
capsaicin (CAP) in the absence or presence of forskolin, as
indicated. An asterisk indicates a significant difference
from basal release, and a cross indicates a significant
difference from capsaicin-stimulated release using an analysis of
variance with Neuman-Keuls as post hoc test
(p < 0.05).
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 |
DISCUSSION |
Our results using RT-PCR and immunoblotting extend our
understanding of which prostaglandin receptor subtypes are expressed in
sensory neurons. Previous studies using in situ
hybridization revealed mRNA to EP1, EP3, EP4, and IP receptors in
dorsal root ganglia neurons (32, 33). Furthermore, the major
distribution of PGE2 binding sites and of EP3-like
immunoreactivity in the spinal cord is in lamina I and II of the dorsal
horn (34, 35), and this coincides with the location of the terminal
endings of small diameter sensory neurons that conduct pain signals
(36). These studies, however, did not examine the localization of the EP2 receptor; nor were attempts made to define which splice variants of
EP3 are found in sensory neurons. Our findings demonstrate that sensory
neurons are capable of expressing EP1, EP2, EP3C, EP4, and IP
receptors. We could not detect any signal for either EP3A or EP3B
receptor mRNAs using PCR. We did not examine EP3D receptor
expression in sensory neurons, since this receptor subtype has not been
cloned from mice or rats. Because we could detect bands for the EP3A
and EP3B receptor when we use cDNA from rat kidney and the same PCR
protocol, we are confident that the lack of detection of EP3A and EP3B
mRNA in sensory neurons is not secondary to methodological
problems. While the sizes of the EP1, EP2, EP3, and IP receptors in rat
sensory neurons were identical to EP receptors from other species
(28-30, 58, 59), the size of the EP4 receptor was similar to the
porcine EP4 receptor (30) but not human EP4 receptor (59). This
variation in size may reflect glycosylation of the receptor or perhaps
species variation between rat and human EP4 receptors.
Because multiple EP receptor subtypes are present in sensory neurons,
it is important to define which subtypes mediate the biological actions
of PGE2. In the case of sensory neurons, exposure to
PGE2 does not directly activate the neurons but renders
them more sensitive to external stimuli. This sensitization results in
a reduced threshold for action potential firing (16), an increase in
cell firing for a given stimulus (19, 37), and an increase in the
amount of neurotransmitter release by a stimulating agent (20, 38). We
previously have shown that the PGE2-induced augmentation of
peptide release is mediated by activation of the cAMP transduction
cascade (18). In a similar manner, enhanced pain sensitivity
(hyperalgesia) and the increase in firing of sensory neurons by
PGE2 also require an increase in cAMP (39-41). Thus, the
EP receptor subtypes that potentially mediate sensitivity because they
have been linked to an increase in cAMP are the EP2 (5, 6), EP3C (9,
11, 60), and/or EP4 (5). The ability of PGE2 to increase
cAMP levels and to augment peptide release is blocked by reducing
expression of the EP3C and EP4 receptors using antisense directed at
mRNA for these subtypes. In contrast, antisense to the EP2 receptor
or the combination of antisense to the EP2 and EP4 receptor does not
alter the effects of PGE2. These results strongly suggest
that the EP3C and EP4 receptors either cooperatively or independently
mediate PGE2-induced sensitization of sensory neurons.
We chose to use antisense to reduce expression of E-type receptors
because of the lack of availability of selective antagonists to the
receptor subtypes (1, 2). We selected exposure to 1 µM
antisense oligonucleotide for a total of 48 h, with a change of
medium and oligonucleotide at 24 h, since previous studies have demonstrated a significant loss of protein activity with this
protocol (44-46) and our concentration-response results indicated that
this protocol achieved significant inhibition of EP receptor expression. Based on our findings, we conclude that antisense oligonucleotides are useful to selectively reduce receptor expression for a number of reasons. First, using antibodies against the EP2, EP3,
and EP4 receptors, we showed that antisense but not missense treatment
significantly reduced protein expression. In addition, exposure to
antisense does not affect the expression of the housekeeping protein,
actin, in sensory neurons. Second, treatment with individual antisense
or missense oligonucleotides or combinations of missense oligonucleotides did not alter the ability of PGE2 to
increase cAMP or augment peptide release, suggesting that the effects
of reducing EP3C and EP4 expression are not mediated by a lack of specificity or toxicity of the oligonucleotides. Finally, although the
combination of antisense to EP3C and EP4 receptors abolished the
effects of PGE2, this treatment did not block the
forskolin-stimulated production of cAMP or the forskolin-induced
augmentation of peptide release from sensory neurons, indicating that
the cAMP-dependent sensitization of sensory neurons (18)
was not affected.
Our findings of the primary role of the EP3C and EP4 receptor in
sensitization do not agree with previous work that suggested that the
EP2 and EP3A receptor are involved in PGE2-induced enhanced excitability of sensory neurons (42, 43). One limitation of those
studies, however, was that the concentrations of EP agonists used were
10-100-fold greater than the Ki of the drug for the
receptor. Thus, the selectivity of the prostaglandin agonist for a
specific EP receptor that was used to identify receptor subtypes
involved in hypersensitivity is questionable. Furthermore, since the
authors' identification of the EP3A receptor was based on the
pharmacological profile of EP receptor agonists (42, 43) rather than on
the mRNA or immunological detection of the receptor and since the
EP agonist used in that study can activate the EP3C and EP4 receptor
subtypes (6, 11, 61), it is possible that these receptors and not the
EP3A were being activated.
It is interesting that exposure to a 100 nM concentration
of the putative EP1/EP3 agonist, sulprostone, or the EP2/EP4 agonist 1-OH-PGE1 did not enhance icAMP content in sensory neurons.
This lack of effect of sulprostone confirms previous work by Smith et al. (62) using adult sensory neurons. It is possible that higher concentrations of sulprostone or 1-OH-PGE1 might
increase icAMP content; however, the lack of specificity of these
agents at higher concentrations could cloud interpretation of results.
In contrast, 1 µM butaprost did increase cAMP, suggesting
that the EP2 receptor might mediate the sensitizing actions of
PGE2, and this is inconsistent with the results from
antisense studies. The findings with butaprost could be explained by a
lack of specificity of the agent. Although antisense to EP2 reduces the
effects of butaprost on cAMP, it does not completely block the action.
Furthermore, antisense to both the EP3C and the EP4 receptors also
significantly reduces the effects of butaprost, although these
antisenses do not reduce EP2 expression. Thus, the effect elicited by 1 µM butaprost is not restricted to the EP2 receptor and
may be mediated by activation of other prostanoid receptors. In
addition, butaprost at micromolar concentrations binds to the mouse IP
receptor (57), and IP receptor agonists increase icAMP content in
sensory neurons (18, 62). Since IP receptor expression was not affected
by antisense treatment to EP3C and EP4, there are sufficient IP
receptors available to potentially mediate the actions of butaprost. It
also is possible that butaprost, but not PGE2, activates
the EP2 receptor in the isolated sensory neurons. Exposure to antisense
against the EP2 receptor significantly decreased EP2 expression in
isolated neurons, without affecting EP3 or EP4 expression and without
affecting PGE2-induced icAMP production, suggesting that
the EP2 receptor is not critical to the actions of PGE2 and
is only partially active in the butaprost-stimulated cAMP production.
Further studies are warranted to ascertain the role of IP receptors in
prostanoid sensitization and to determine whether various
pharmacological agents act at specific receptors on sensory neurons.
Overall, our results indicate that the use of antisense
oligonucleotides is a more direct approach to study the EP receptor subtypes involved in sensitization of sensory neurons than the use of
EP receptor agonists. Antisense oligonucleotides abolish the expression
and activity of a specific EP receptor subtype (Fig. 4) without
affecting expression of other EP receptor subtypes or the IP receptor.
Also, any potential lack of selectivity or toxicity of oligonucleotides
can be assessed, and adequate control experiments can be performed to
assure the specificity of the antisense oligonucleotide (Figs. 3 and 6
and Table I). Finally, antisense oligonucleotides diminish EP receptor
expression in a dose-response manner, whereas the potential lack of
specificity that can occur with higher concentrations of EP agonists
can cloud interpretation of the results (1, 2). Thus, the use of
antisense oligonucleotides provides a powerful new tool to examine the
subtypes of EP receptors that mediate a physiological process.
Since two subtypes of PGE2 receptor, the EP3C and EP4
subtype, either cooperatively or independently mediate the
PGE2-induced sensitization of sensory neurons, the question
remains as to whether these EP receptors co-exist on the same sensory
neurons. Our primary cultures contain a heterogeneous population of
sensory neurons that differ in neuronal size, capsaicin sensitivity,
and neuropeptide content (47). Thus, it is possible that different
receptor subtypes could be expressed on different subpopulations of
neurons and thus subserve different and, as yet, unknown functions.
Indeed, Oida et al. (33) report that the EP1 receptor
mRNA was expressed in ~30%, the EP3 mRNA in ~50%, the EP4
in ~20%, and the IP receptor mRNA in ~40% of the dorsal root
ganglia neurons. It is clear from the current work, however,
that both EP3C and EP4 receptors are functionally expressed on a subset
of capsaicin-sensitive sensory neurons, since this was the agent we
used to stimulate peptide release.
Another possibility is that PGE2 receptor subtypes are
expressed in different densities on a given neuron, and the predominant receptor mediates the biological response. Multiple subtypes of the
PGE2 receptor are expressed in cilary epithelial cells
(48), osteoblastic cells (49), kerotinocytes (50), fibroblasts (23), and platelets (51). Furthermore, human Jurkat cells express both EP2
and EP4 receptors; however, the expression of EP2 receptor is
~100-fold lower than that of EP4 receptors (52). In these cells, the
EP4 receptors are the primary receptor subtype mediating the
PGE2-induced increase in cAMP, whereas EP2 receptors appear to have little or no functional role in PGE2-induced cAMP
production (52). An analogous situation might exist in sensory neurons. It would be interesting to determine whether the EP3C and EP4 receptor
subtypes are equally expressed in cultures of sensory neurons and
whether the expression of EP1 and EP2 in sensory neurons is of a
similar magnitude.
Further studies are warranted to ascertain whether EP receptor subtypes
are differentially expressed in the same neuron or exist in separate
subpopulations of sensory neurons. However, if multiple receptors
mediate sensitization of sensory neurons in situ, then
attempts to inhibit a single receptor to block the inflammatory actions
of PGE2 might be ineffective. Furthermore, although
transgenic mice with knockouts of single EP receptor subtype have been
developed (53-56), the redundancy of EP receptor functions, which we
have reported in the present study, could limit the value of these
single receptor transgenic models of nociceptive transmission. Future
studies using double knockout mice might be more appropriate in
studying the mechanisms of pain and inflammation.