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The Journal of Neuroscience, March 1, 2001, 21(5):1750-1756
Direct Activation of Rat Spinal Dorsal Horn Neurons by
Prostaglandin E2
Hiroshi
Baba,
Tatsuro
Kohno,
Kimberly A.
Moore, and
Clifford J.
Woolf
Neural Plasticity Research Group, Department of Anesthesia and
Critical Care, Massachusetts General Hospital and Harvard Medical
School, Charlestown, Massachusetts 02129
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ABSTRACT |
Whole-cell patch-clamp and intracellular recording techniques have
been used to study the action of prostaglandin E2 (PGE2) on neurons in
adult rat transverse spinal cord slices. Bath-applied PGE2 (1-20
µM) induced an inward current or membrane depolarization in the majority of deep dorsal horn neurons (laminas III-VI; 83 of 139 cells), but only in a minority of lamina II neurons (6 of 53 cells). PGE2 alone never elicited spontaneous action potentials; however, it did convert subthreshold EPSPs to suprathreshold, leading
to action potential generation. PGE2-induced inward currents were
unaffected by perfusion with either a Ca2+-free/high
Mg2+ (5 mM) solution or
tetrodotoxin (1 µM), indicating a direct
postsynaptic action. Both 17-phenyl trinor prostaglandin E2 (an EP1
agonist) and sulprostone (an EP3 agonist) had little effect on membrane current, whereas butaprost methyl ester (an EP2 agonist) mimicked the
effect of PGE2. Depolarizing responses to PGE2 were associated with a
decrease in input resistance, and the amplitude of inward current was
decreased as the holding potential was depolarized. PGE2-induced inward
currents were reduced by substitution of extracellular Na+ with
N-methyl-D-glucamine and inhibited by
flufenamic acid (50-200 µM), which is compatible with
activation of a nonselective cation channel. These results suggest that
PGE2, acting via an EP2-like receptor, directly depolarizes spinal
neurons. Moreover, these findings imply an involvement of spinal
cord-generated prostanoids in modulating sensory processing through an
alteration in dorsal horn neuronal excitability.
Key words:
inflammation; central sensitization; prostaglandin E2; EP2 receptor; deep dorsal horn; allodynia; hyperalgesia
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INTRODUCTION |
After peripheral tissue injury or
inflammation, exaggerated pain behavior occurs that includes
hyperalgesia, an increased responsiveness to noxious stimuli, and
allodynia, pain elicited in response to normally innocuous stimuli.
Prostaglandins (PGs) synthesized by the inducible isoform of
cyclooxygenase (COX-2) at the site of tissue injury sensitize
peripheral nociceptors through activation of EP receptors on
peripheral nerve terminals (Kumazawa et al., 1993 ). This peripheral
sensitizing action contributes to heightened sensitivity at the site of
tissue injury (primary hyperalgesia) (Ohuchi et al., 1976; Baccaglini
and Hogan, 1983). Therefore, the analgesic effects of COX inhibitors
[i.e., nonsteroidal anti-inflammatory drugs (NSAIDs)] have long been
thought to be mediated primarily through inhibition of PG generation in
the periphery (Vinegar et al., 1976). Recently, however, evidence has
accumulated to indicate that PGE2 is also produced in the spinal cord
after tissue injury (Yang et al., 1996; Dirig and Yaksh, 1999; Samad et
al., 2001). Furthermore, behavioral studies suggest that PGE2 may
facilitate nociceptive transmission in the spinal cord (Uda et al.,
1990; Minami et al., 1994), contributing to central sensitization, a
central amplification of sensory outflow from the spinal cord that is
responsible for the spread of sensitivity beyond the site of injury
(Woolf and King, 1990).
Constitutive levels of COX-2 in the spinal cord are low, but peripheral
inflammation upregulates this enzyme (Beiche et al., 1996, 1998;
Ichitani et al., 1997), leading to production of PGE2 in the spinal
cord (Yang et al., 1996; Dirig and Yaksh, 1999). Intrathecally
administered PGE2 evokes hyperalgesia and allodynia (Uda et al., 1990;
Minami et al., 1994), and both the behavioral hyperalgesia and the
increase in spinal PGE2 concentration induced by peripheral
inflammation are attenuated by intrathecally administered NSAIDs
(Malmberg and Yaksh, 1992, 1995; Yamamoto and Nozaki-Taguchi, 1997).
Intrathecal NSAIDs also reduce noxious stimulus-induced or
peripheral inflammation-induced dorsal horn neuron hyperexcitability (Chapman and Dickenson, 1992; Pitcher and Henry, 1999). Although these
studies collectively suggest a nociceptive role for PGE2 produced by
COX in the spinal cord, it is uncertain what is responsible for its
production, where it is produced, and where and how it acts.
Autoradiographic studies indicate that the highest density of spinal
PGE2 binding sites is in lamina II, substantia gelatinosa (SG).
PGE2 binding in lamina II is reduced, but not eliminated, by dorsal
rhizotomy (Matsumura et al., 1995), suggesting that receptor sites are
located on both presynaptic terminals of unmyelinated nociceptive
fibers and postsynaptic dorsal horn neurons. Therefore, PGE2 may act
presynaptically to facilitate neurotransmitter release (Nicol et al.,
1992; Hingtgen et al., 1995; Vasko, 1995) and postsynaptically to
directly excite dorsal horn neurons. Here, we demonstrate that PGE2
does directly activate a subpopulation of spinal dorsal horn neurons.
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MATERIALS AND METHODS |
The methods for preparing thick adult rat spinal cord slices, as
well as intracellular and blind whole-cell patch-clamp recording techniques, have been described in detail previously (Yoshimura and
Jessell, 1989; Yoshimura and Nishi, 1993; Baba et al., 1999). Briefly,
adult male rats (7-10 weeks old; 250-350 gm) were anesthetized with
urethane (1.5-2.0 gm/kg, i.p.), and the lumbosacral spinal cord was
removed. The isolated spinal cord was then placed in preoxygenated
ice-cold Krebs' solution (2-4°C). After removal of the dura mater,
all ventral and dorsal roots were cut, and the pia-arachnoid was
removed. In some experiments, a dorsal root (L4) was preserved to
permit stimulation of primary afferent fibers. The spinal cord was
placed in a shallow groove formed in an agar block, glued to the bottom
of a microslicer stage with cyanoacrylate adhesive, and immersed in
ice-cold Krebs' solution. A 500- to 600-µm-thick transverse slice
was cut on a vibrating microslicer (DTK1500; Dosaka Co. Ltd., Kyoto,
Japan), placed on a nylon mesh in the recording chamber, and held in
place by a titanium electron microscopy grid supported by a silver-wire
loop. The slice was perfused with Krebs' solution (15 ml/min)
saturated with 95% O2 and 5%
CO2 at 36-37°C. The Krebs' solution contained
(in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose. To examine the effects of
removing extracellular sodium on PGE2-induced currents, 117 mM NaCl was replaced with 117 mM
n-methyl-D-glucamine (NMDG).
Blind whole-cell patch-clamp and intracellular techniques were used to
record from dorsal horn neurons. For whole-cell patch-clamp recording,
the pipette solution contained (in mM): 135 potassium gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 ATP-Mg salt, and 0.5 Na-GTP. The resistance of a typical patch pipette was 5-10 M .
Membrane currents (patch-clamp recording) were amplified with an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA) in
voltage-clamp mode; usually a 75% series resistance compensation was
introduced. Unless otherwise indicated, membrane currents were recorded
at a holding potential of  70 mV. For intracellular recordings,
membrane potentials were amplified with an Axoclamp 2A amplifier (Axon
Instruments). The resistance of a typical "sharp" electrode for
intracellular recording was 150-200 M when filled with 4 M potassium acetate. Signals were filtered at 2 kHz and digitized at 5 kHz. Data were analyzed using pCLAMP 6 software (Axon
Instruments).
Recording electrodes were positioned in the dorsal horn under direct
visual control. In the adult spinal cord, lamina II [substantia gelatinosa (SG)] is readily identifiable as a distinct translucent region. When recording from SG neurons, the recording electrode was
positioned in the middle third of the SG. We found that when an
electrode is targeted to lamina II and neurons are labeled with
neurobiotin, the recorded neuron is invariably located in the targeted
lamina. This suggests that recording from dendrites or axons of neurons
located in other laminas is extremely rare (Baba et al., 1999). The
border between the SG and lamina III is clear, but the borders of
laminas IV, V, and VI are not distinguishable; therefore, we dealt with
laminas III-VI as one group (classified as deep dorsal horn neurons in
Table 1). Blind recordings from lamina I were not attempted because of
the technical difficulties that arise from the orientation of the neurons.
Orthodromic stimulation of the dorsal root (L4) was performed with a
suction electrode and a constant-current stimulator (Neurolog). The
threshold stimulation intensity and duration of A -fiber (~10 µA,
0.05 msec), A -fiber (~25 µA, 0.05 msec), and C-fiber
(~200 µA, 0.5 msec) for this suction electrode have been
established previously (Baba et al., 1999). Identification of
EPSCs/EPSPs as monosynaptic was based on a constant latency with graded
intensity and high-frequency repetitive stimulation (20 Hz).
Polysynaptic EPSCs/EPSPs, in contrast, displayed variable latencies
with such stimulation protocols.
Drugs were applied by exchanging the perfusion solution with one
containing a known drug concentration, without altering the perfusion
rate and the temperature. Drugs used were prostaglandin E2 (a gift from
Ono Pharmaceutical, Osaka, Japan), flufenamic acid (FFA; Sigma, St.
Louis, MO), NMDG (Sigma), 17-phenyl trinor prostaglandin E2 (Cayman
Chemical, Ann Arbor, MI), sulprostone (Cayman Chemical),
19(R)-hydroxy prostaglandin E2 (Cayman Chemical), butaprost methyl ester (Cayman Chemical), and tetrodotoxin (TTX; Sigma). Prostanoid stock solutions were prepared in DMSO. Stocks were
diluted 1:2800 in Krebs' solution, resulting in a final DMSO concentration of <0.1%. This concentration of DMSO produces no measurable effect on dorsal horn neurons. Data are presented as the
mean ± SD.
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RESULTS |
PGE2-induced depolarization and inward current
Bath-applied PGE2 (1-20 µM) induced a membrane
depolarization (measured by intracellular recording) or an inward
current (measured by whole-cell patch-clamp recording) in most of the
deep dorsal horn neurons (83 of 139 neurons in laminas III-VI) (Fig.
1A-C). In contrast,
PGF2 and PGD2 were without effect (n = 5;
Fig. 1C). The PGE2-induced membrane depolarization in deep
dorsal horn neurons averaged 8 ± 4 mV (laminas III-VI; PGE2, 10 µM; n = 18), whereas the inward
current averaged 21 ± 16 pA (laminas III-VI; PGE2, 10 µM; n = 65) (Table
1). In a subset of experiments, neurons were classified on the basis of primary afferent input (Fig.
1B). PGE2 responses were significantly greater in
neurons with both A- and C-fiber inputs than in neurons receiving
solely A-fiber inputs (38 ± 23 pA, n = 8, vs
16 ± 10 pA, n = 9; p = 0.02, unpaired t test). In contrast, the vast majority of lamina
II neurons tested did not respond to PGE2 (10 µM), with only 6 of 53 SG neurons (11%)
showing a PGE2-induced inward current or depolarization. The amplitude
of PGE2 (10 µM)-induced currents in those SG
neurons that responded (5 ± 2 pA; n = 5) was
significantly smaller than in deep dorsal horn neurons (21 ± 16 pA, n = 65; p = 0.023, unpaired t test; Table 1).
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Figure 1.
Effect of PGE2 on membrane properties of deep
dorsal horn neurons. A, Depolarizing response produced
by bath application of PGE2 (10 µM) recorded
intracellularly in current-clamp mode in the presence of TTX (1 µM). Downward deflections are electrotonic voltage
transients elicited by hyperpolarizing current pulses (amplitude, 0.15 nA; duration, 400 msec) to measure membrane input resistance
(Rin). The PGE2-induced
depolarization was associated with a decreased
Rin. When the neuron was manually clamped
(DC) to the resting membrane potential, the reduction in
Rin was unaffected. Therefore, the decreased
Rin was not caused by activation of
voltage-gated channels. B, Blind whole-cell patch-clamp
recording of a PGE2 (5 µM)-induced inward current
recorded in the presence of TTX (1 µM) from a neuron
voltage clamped to 70 mV. This neuron was recorded from a slice with
an attached dorsal root, permitting determination of the type of
afferent input. Neurons with both A- and C-fiber inputs
(bottom) responded to PGE2 with larger inward currents
than neurons with purely A-fiber input. C, Both PGF2
(10 µM) and PGD2 (10 µM) were without
effect in this PGE2 (10 µM)-responsive neuron.
D, Intracellular recording from a deep dorsal horn
neuron. Top, Bath-applied PGE2 (10 µM)
elicited a membrane depolarization. At the resting membrane potential,
dorsal root stimulation at A-fiber intensity (30 µA, 0.05 msec, 0.5 Hz) elicited subthreshold EPSPs. During the PGE2-induced
depolarization, evoked EPSPs reached action potential threshold. Action
potentials are indicated by dots. When the neuron was
manually clamped (DC) to the resting membrane potential,
action potentials disappeared. Bottom, EPSPs and action
potentials are shown on an expanded time scale. Note that the
amplitudes of EPSPs during DC are not significantly different from
control, indicating that the generation of action potentials is not
caused by the augmentation of transmitter release from presynaptic
terminals. Three consecutive traces are superimposed in
each stimulating condition.
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To determine whether the PGE2-induced depolarization affected the
action potential generation, dorsal root-evoked responses were examined
in deep dorsal horn neurons that exhibited PGE2 responses. In all six
neurons tested, dorsal root stimulation at A-fiber intensity evoked
either monosynaptic EPSPs or polysynaptic EPSPs, or both.
Although PGE2 alone never elicited spontaneous action potentials in
these neurons, it converted subthreshold inputs to suprathreshold and
led to action potential generation (Fig. 1D). These
data suggest that the PGE2-induced depolarization increased the
probability that subthreshold EPSPs in deep dorsal horn neurons reached
the action potential threshold.
Several lines of evidence suggest that PGE2 can facilitate excitatory
transmitter (e.g., glutamate, substance-P, etc.) release, particularly
from primary afferent C-fiber terminals (Nicol et al., 1992; Hingtgen
et al., 1995; Vasko, 1995). Therefore, slow inward currents elicited by
PGE2 might result from an indirect excitatory action on central primary
afferent terminals (presynaptic action). To test the possibility of
such an effect, the effect of PGE2 on deep dorsal horn neurons was
examined either under synaptic blockade or in the presence of TTX to
block action potentials. TTX (1 µM; n = 10) did not significantly modify the depolarizing or inward
current-generating effects of PGE2 (Fig.
1A,B). Similarly, in
Ca2+-free/high
Mg2+ (5 mM) Krebs'
solution, PGE2-induced inward currents were also fully preserved
(n = 7; Fig.
2A), although primary
afferent stimulation-evoked monosynaptic EPSCs were completely
abolished (Fig. 2B). The frequency of spontaneous
miniature EPSCs (mEPSCs) in those deep dorsal horn neurons that
showed an inward current in response to PGE2 was also unaffected by
PGE2 (102 ± 12% of control; p = 0.68;
n = 10; paired t test; Fig.
2C).
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Figure 2.
PGE2 acts postsynaptically. A,
Whole-cell patch-clamp recording of a PGE2 (5 µM)-evoked
inward current in a deep dorsal horn neuron with only A-fiber input.
The PGE2-induced inward current was not blocked by perfusion with the
Ca2+-free/high Mg2+ (5 mM) Krebs' solution. B, In contrast, dorsal
root-evoked monosynaptic EPSCs were completely abolished by perfusion
with the Ca2+-free/high Mg2+
solution. Traces in A and
B are from the same neuron. C, PGE2 did
not affect the frequency of miniature EPSCs. The frequencies of
miniature EPSCs before and during application of PGE2 were 46.2 and
44.3 Hz, respectively. This neuron responded to PGE2 with an inward
current (see Fig. 1B).
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In addition, we tested whether PGE2 facilitated A-fiber- and
C-fiber-mediated excitatory transmission in the SG in which many A-
and C-fibers make direct synaptic connections. PGE2 (10 µM) had no significant effect on the amplitude of
monosynaptic A-fiber-evoked (237 ± 164 vs 239 ± 151 pA;
n = 9; p = 0.79) or C-fiber-evoked
(355 ± 83 vs 350 ± 89 pA; n = 8;
p = 0.78) EPSCs (Fig.
3A,B).
Furthermore, the frequency of miniature EPSCs in SG neurons was not
significantly affected by PGE2 (10 µM;
n = 15; p = 0.23; Fig.
3C,D).
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Figure 3.
PGE2 has no effect on excitatory transmitter
release in the SG (lamina II). A, The amplitude of
A-fiber (top traces) and C-fiber (bottom
traces)-evoked monosynaptic EPSCs was not affected by PGE2 (10 µM). Five consecutive traces are
superimposed for each condition. They were averaged, and the difference
between baseline and peak current was measured. B,
Summary of the PGE2 (10 µM) effects on the amplitude of
dorsal root-evoked EPSCs. PGE2 did not significantly alter the
amplitude of either A- or C-fiber-evoked EPSCs in the SG (A-fiber
EPSC, p = 0.79, n = 9; C-fiber
EPSC, p = 0.78, n = 8; paired
t test). The amplitudes of evoked EPSCs were measured
5-10 min after the start of PGE2 application. C,
Miniature EPSCs recorded from a SG neuron in the presence of TTX (1 µM). The frequencies of mEPSCs were 22.6 and 22.2 Hz
before and during application of PGE2, respectively. D,
The effect of PGE2 (10 µM) on the frequency of mEPSCs.
PGE2 did not significantly increase the frequency of mEPSCs in 14 of 15 SG neurons studied (p = 0.23, paired
t test). The frequency of mEPSCs was counted 5-10 min
after the start of PGE2 application. All data were recorded at 70
mV.
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An EP2-like receptor mediates the PGE2-induced inward current
The known EP receptors have been divided into four subtypes, EP1,
EP2, EP3, and EP4 (Coleman et al., 1994). Because selective EP receptor
antagonists are not available, we used a battery of agonists to
determine which EP receptor subtype mediates the PGE2-induced inward
current. A nonselective EP receptor agonist,
19(R)-hydroxy prostaglandin E2 (3-10
µM) (Boie et al., 1997), and a relatively selective EP2 receptor agonist, butaprost methyl ester (3-10
µM) (Boie et al., 1997), both evoked inward
currents in a concentration-dependent fashion
[19(R)-hydroxy PGE2, 17 ± 3 pA at 10 µM, n = 5 of 7; butaprost methyl ester, 14 ± 7 pA, n = 4 of 6; Fig.
4A-C]. The
EC50 of the butaprost methyl ester response was
3.1 µM, consistent with its binding affinity
for the EP2 receptor (2.6 µM) (Boie et al.,
1997). In contrast, both an EP1 agonist, 17-phenyl trinor prostaglandin E2 (0.3-10 µM; n = 6), and an
EP3 agonist, sulprostone (0.3-10 µM;
n = 5), had little effect in neurons that responded to
PGE2 (10 µM; Fig.
4A,C). These results suggest that
the PGE2-induced inward current is mediated by an EP receptor with an
EP2-like profile.
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Figure 4.
Effects of EP receptor agonists on membrane
currents in deep dorsal horn neurons. A, An EP1 receptor
agonist, 17-phenyl trinor prostaglandin E2 (10 µM), and
an EP3 receptor agonist, sulprostone (10 µM), had no
effect on membrane currents in neurons that responded to the
nonselective EP receptor agonist, 19(R)-hydroxy
prostaglandin E2 (10 µM). B, The selective
EP2 receptor agonist, butaprost methyl ester (10 µM),
induced an inward current similar to that produced by PGE2 (10 µM). C, Comparison of the effects of the
different EP agonists. Points represent the mean ± SD of the percentage maximum response to PGE2 (10 µM).
n = 3-6 in each group. EC50 values are
1.5 µM for PGE2, 2.3 µM for
19(R)-hydroxy prostaglandin E2, and 3.1 µM for butaprost methyl ester.
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PGE2 activation of a cation conductance
The PGE2-induced depolarization in deep dorsal horn neurons was
associated with a decrease in input resistance (13 ± 5%;
n = 5; Fig. 1A), indicating that the
opening of membrane channels, rather than the closing of potassium
channels, underlies the PGE2 response. Additionally, when the
holding potential was depolarized (from 90 to 50 mV;
n = 4; Fig.
5A), inward currents decreased in amplitude. Therefore, it is unlikely that activation of
Cl channels (ECl = 70 mV) contributes to the PGE2 response. In distal colon cells, PGE2
activates a nonselective cation conductance (Siemer and Gogelein,
1992). To determine whether a similar mechanism might exist in dorsal
horn neurons, we examined the effects of FFA (50-200
µM), a nonselective blocker of cation channels
(Gogelein et al., 1990). FFA alone had no effect on membrane current,
but it reversibly blocked PGE2-induced inward currents in deep dorsal horn neurons (n = 8; Fig.
5B,C). The sodium permeability of
the PGE2-activated channel was also investigated by replacing
extracellular sodium with a nonpermeable cation, NMDG (Fig.
5D). PGE2 was initially applied in normal Krebs' solution;
then the low sodium (NMDG) solution was exchanged for normal
extracellular solution. PGE2-induced inward currents were immediately
reduced to nearly baseline levels by the low sodium solution in a fully
reversible fashion in all cells tested (n = 4).
Baseline membrane currents were not significantly affected by switching
to the low-sodium solution.
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Figure 5.
Effects of membrane potential, flufenamic acid
(FFA), and
N-methyl-D-glucamine (NMDG)
on PGE2-induced inward currents. A, This deep dorsal
horn neuron was voltage clamped to different holding potentials, and
PGE2 (10 µM, 60 sec)-induced inward currents were
recorded. Note that responses become smaller as the membrane
potential is depolarized. HP, Holding potential.
B, Bath-applied PGE2 (5 µM) elicited an
inward current in another neuron, as recorded by the whole-cell
patch-clamp technique. FFA (50 µM) partially blocked the
PGE2-induced inward current. Increasing the concentration of FFA to 100 µM almost completely blocked the PGE2-induced current, in
a reversible fashion. C, Whole-cell patch-clamp
recording reveals that application of FFA (100 µM) during
the PGE2 (5 µM)-induced inward current reversed the
effect of PGE2. D, Effect of decreasing extracellular
Na+ on PGE2-induced inward currents. In normal
Krebs' solution, bath-applied PGE2 (10 µM) induced an
inward current. Changing the perfusion solution to a low
Na+ solution, in which Na+ was
replaced with NMDG+ (a membrane impermeable
cation), reversed the PGE2-induced inward current nearly to control
values. Returning to normal Krebs' solution restored the PGE2-induced
inward current. The amplitudes of spontaneous EPSCs are truncated.
Neurons were voltage clamped to 70 mV in
B-D.
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DISCUSSION |
We have found that the prostanoid PGE2 induces an inward current
or membrane depolarization in many deep dorsal horn neurons from thick
adult rat spinal cord slices, whereas PGF2 and PGD2 have no effect.
PGE2 acts directly on the recorded neurons because the inward current
is preserved under conditions in which synaptic transmission is
blocked. The frequency of spontaneous EPSCs is not affected by PGE2
during the inward current, further suggesting a direct postsynaptic
effect. PGE2-induced inward currents were associated with a reduction
in input resistance, and they decreased in amplitude when the holding
potential was depolarized (from 90 to 50 mV). Therefore, it is
unlikely that the PGE2 response is mediated by
K+ or Cl
channels that have hyperpolarized equilibrium potentials
(K+ = 90 mV;
Cl = 70 mV). Together with the
reduction in the PGE2 current by flufenamic acid and the effect of
replacing extracellular sodium, this suggests that the PGE2-induced
depolarization may be mediated through activation of a nonselective
cation channel.
Although intrathecally administered PGE2 produces allodynia and
hyperalgesia (Uda et al., 1990; Minami et al., 1994), the site and
mechanism of action have not yet been elucidated. Autoradiographic studies demonstrate that the most intense binding of PGE2 is in the
superficial dorsal horn. Intense PGE2 binding is reduced after dorsal
rhizotomy (Matsumura et al., 1995), suggesting that PGE2 binds to
receptor sites on the central terminals of C-fibers. Indeed, some
studies suggest that PGE2 enhances glutamate release from primary
afferent C-fiber terminals (Malmberg et al., 1995; Ferreira and
Lorenzetti, 1996). PGE2 also stimulates neuropeptide release from
cultured embryonic DRG neurons (Nicol et al., 1992; Vasko et al., 1994;
Hingtgen et al., 1995) and spinal cord slices (Andreeva and Rang, 1993;
Vasko, 1995; Southall et al., 1998). Surprisingly, in the present
study, we failed to observe augmentation of identified C-fiber-evoked
glutamatergic EPSCs or an increase in the frequency of miniature EPSCs.
In general, neuropeptide release requires more intense excitation of
presynaptic terminals than is required to elicit glutamate release.
Therefore, facilitation of neuropeptide release (such as substance-P
and/or calcitonin gene-related peptide) without affecting
glutamate release is unlikely, unless PGE2 acts specifically to affect
release from dense-core vesicles. Nonetheless, we cannot exclude a
presynaptic PGE2 effect on neuropeptide-containing C-fibers that
synapse on lamina I neurons. Additional studies are required to clarify
which EP receptors are located on the central terminals of primary
afferents and what their role is in regulating transmitter release.
What is clear is that PGE2 does exert a direct postsynaptic excitatory action on deep dorsal horn neurons.
The magnitude of the PGE2 depolarization in deep dorsal horn neurons is
relatively small (5-15 mV); although insufficient to elicit
spontaneous spike firing directly, it increases the probability that
primary afferent-evoked EPSPs will reach the action potential threshold
(approximately 45 mV). In this way, the PGE2-mediated depolarization
could have a major role in modulating dorsal horn transmission (central
sensitization; Fig. 1). Most primary afferent synaptic inputs are
subthreshold, and a small increase in membrane excitability is
sufficient to substantially modify receptive field properties,
including increasing spatial extent and responsiveness, as well as
reducing the threshold (Woolf and King, 1990). Furthermore, membrane
depolarization below the action potential threshold level can generate
plateau potentials via FFA-sensitive nonselective cation channels that
significantly alter the input-output relationship of a proportion of
deep dorsal horn neurons (Morisset and Nagy, 1999).
Determination of the EP receptor subtype involved in PGE2-evoked
responses has been hampered by the absence of antagonists. We have,
instead, used a battery of EP receptor agonists. Although the agonists
used in this study are not 100% selective for the respective EP
receptors, 17-phenyl trinor PGE2 (EP1  EP3) and sulprostone (EP3
EP1) have much higher affinity for EP1 and EP3 receptors than for EP2
or EP4 receptors (Minami et al., 1994). In contrast, butaprost methyl
ester is relatively selective for EP2 receptors (Coleman et al., 1994;
Boie et al., 1997). We have shown that, even at high concentrations,
17-phenyl trinor PGE2 and sulprostone did not mimic PGE2 action, but
butaprost methyl ester did. Therefore, it is likely that the direct
effects of PGE2 on deep dorsal horn neurons were mediated by an
EP2-like receptor.
EP1 and EP2/EP4 receptors are coupled to
Ca2+ mobilization and stimulation of
adenylate cyclase (AC-cAMP-PKA cascade), respectively (Coleman
et al., 1994). In contrast, depending on which splice variant is
present, EP3 receptors can activate or inhibit adenylate cyclase or
mobilize intracellular calcium (Narumiya et al., 1999). Interestingly,
the thermal hypersensitivity induced by intrathecal PGE2 is reduced in
cAMP-dependent protein kinase knock-out mice (PKA mutant mice)
(Malmberg et al., 1997), in keeping with an action on EP2, EP3, or EP4
receptors. Other behavioral studies also suggest that EP2 and EP3
receptor activation elicits hyperalgesia, whereas EP1 receptor
activation results in allodynia (Minami et al., 1994). Therefore, the
direct depolarization of deep dorsal horn neurons described here may
contribute to some of the behavioral effects of PGE2 that have been
described previously.
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FOOTNOTES |
Received March 31, 2000; revised Dec. 7, 2000; accepted Dec. 18, 2000.
This work was supported by Human Frontier Science Program Grant RG73/96
and National Institutes of Health Grant NS38253-01. Dr. Baba was also
supported by the Ministry of Education, Science, Sports and Culture of
Japan (Niigata University, Niigata, Japan). We thank Ono Pharmaceutical
Co. (Osaka, Japan) for the gift of PGE2.
Correspondence should be addressed to Dr. Hiroshi Baba, Department of
Anesthesiology, Niigata University School of Medicine, 1-757 Asahimachi, Niigata 951-8510, Japan. E-mail:
baba{at}med.niigata-u.ac.jp.
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TOP
ABSTRACT
INTRODUCTION
