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The Journal of Neuroscience, September 1, 1998, 18(17):7008-7014
Nitric Oxide Signaling in Pain and Nociceptor Sensitization
in the Rat
K. O.
Aley,
Gordon
McCarter, and
Jon D.
Levine
Departments of Anatomy, Medicine, and Oral Surgery, Division of
Neuroscience, and National Institutes of Health Pain Center (UCSF),
University of California at San Francisco, San Francisco, California
94143-0452
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ABSTRACT |
We investigated the role of nitric oxide (NO) in inflammatory
hyperalgesia. Coinjection of prostaglandin E2
(PGE2) with the nitric oxide synthase (NOS)
inhibitor
NG-methyl-L-arginine
(L-NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also able to reverse that hyperalgesia. This
suggests that NO contributes to the maintenance of, as well as to the
induction of, PGE2-induced hyperalgesia. Consistent with
the hypothesis that the NO that contributes to PGE2-induced
sensitization of primary afferents is generated in the dorsal root
ganglion (DRG) neurons themselves, L-NMA also inhibited the
PGE2-induced increase in tetrodotoxin-resistant sodium
current in patch-clamp electrophysiological studies of small diameter
DRG neurons in vitro. Although NO, the product of NOS,
often activates guanylyl cyclase, we found that PGE2-induced hyperalgesia was not inhibited by coinjection
of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then tested whether the effect of NO depended on
interaction with the adenylyl cyclase-protein kinase A (PKA) pathway,
which is known to mediate PGE2-induced hyperalgesia. L-NMA inhibited hyperalgesia produced by
8-bromo-cAMP (a stable membrane permeable analog of cAMP) or by
forskolin (an adenylyl cyclase activator). However, L-NMA
did not inhibit hyperalgesia produced by injection of the catalytic
subunit of PKA. Therefore, the contribution of NO to
PGE2-induced hyperalgesia may occur in the cAMP second
messenger pathway at a point before the action of PKA.
We next performed experiments to test whether administration of
exogenous NO precursor or donor could mimic the hyperalgesic effect of
endogenous NO. Intradermal injection of either the NOS substrate
L-arginine or the NO donor 3-(4-morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced hyperalgesia. However, this hyperalgesia differed from PGE2-induced hyperalgesia, because it was
independent of the cAMP second messenger system and blocked by the
guanylyl cyclase inhibitor ODQ. Therefore, although exogenous NO
induces hyperalgesia, it acts by a mechanism different from that by
which endogenous NO facilitates PGE2-induced hyperalgesia.
Consistent with the hypothesis that these mechanisms are distinct, we
found that inhibition of PGE2-induced hyperalgesia caused
by L-NMA could be reversed by a low dose of the NO donor
SIN-1. The following facts suggest that this dose of SIN-1
mimics a permissive effect of basal levels of NO with regard to
PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not
produce hyperalgesia when administered alone, and (2) the effect was
not blocked by ODQ.
In conclusion, we have shown that low levels of NO facilitate
cAMP-dependent PGE2-induced hyperalgesia, whereas higher
levels of NO produce a cGMP-dependent hyperalgesia.
Key words:
hyperalgesia; nitric oxide; pain; primary afferent
nociceptor; prostaglandin E2; protein kinase A; tetrodotoxin-resistant sodium current
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INTRODUCTION |
Tissue injury results in
hyperalgesic pain (tenderness), probably the most common presenting
clinical symptom. This important phenomenon is believed to be
attributable, in great part, to sensitization of primary afferent
nociceptors so that they respond at a lower stimulus intensity and with
greater number of action potentials. Inflammatory mediators have been
implicated in producing this sensitization and hyperalgesia. Of these,
prostaglandins are well established as mediators of mechanical
hyperalgesia in both animals and humans (Collier and Schneider, 1972 ;
Moncada et al., 1975; Ferreira et al., 1978) and of sensitization of
primary afferent nociceptors (Martin et al., 1987; Schaible and
Schmidt, 1988; Davis et al., 1993; Rueff and Dray, 1993). The
inflammatory mediator prostaglandin E2
(PGE2) is thought to act directly on the peripheral terminals of primary afferent nociceptors to produce hyperalgesia (Taiwo and Levine, 1989), to sensitize nociceptors in vitro
(England et al., 1996; Gold et al., 1996a), and to enhance
tetrodotoxin-resistant voltage-gated sodium current (TTX-R
INa). Previous work in our laboratory
suggests that PGE2-induced sensitization of nociceptors is
mediated by the adenylyl cyclase-cAMP-protein kinase A (PKA) second
messenger system. For example, agents that inhibit adenylyl cyclase, as
well as those that inhibit PKA, attenuate PGE2-induced hyperalgesia (Taiwo and Levine, 1989, 1991; Khasar et al., 1995). PGE2 is a key mediator of inflammatory hyperalgesia.
A number of observations suggest that in the periphery nitric oxide
(NO) also acts as a pronociceptive mediator (Moulton, 1996; Robbins and
Grisham, 1997; Wallace and Chin, 1997). Intracutaneous injections of NO
precursors evoke pain in humans (Houlthusen and Arndt, 1994, 1995).
That NO is generated within nociceptors is suggested by these
observations: (1) neuronal nitric oxide synthase-like immunoreactivity
(nNOS-LI) is expressed in small- and medium-diameter dorsal root
ganglion (DRG) neurons in rat and monkey (Zhang et al., 1993; Qian et
al., 1996); (2) NADPH-diaphorase activity (Vanhatalao et al., 1996)
and nNOS-LI (Zhang et al., 1993; Majewski et al., 1995) are colocalized
in some DRG neurons with substance P-LI (SP-LI) and calcitonin
gene-related peptide-LI (CGRP-LI); and (3) NOS-LI is coexpressed
in some DRG neurons with CGRP-LI or SP-LI (Zhang et al., 1993) and is
greatly reduced in DRG neurons in neonatal rats treated with capsaicin
(Ren and Ruda, 1995). NO is considered to play a role in the induction
of nociception, because NOS-LI in lumbar DRG neurons is
increased by noxious irritation of the bladder (Vizzard et al., 1996),
by noxious stimulation with resiniferatoxin (Farkas-Szallasi et al.,
1995; Vizzard et al., 1995), and by induction of neuropathic models of
pain using sciatic nerve transection (Fiallos-Estrada et al., 1993;
Zhang et al., 1993; Beesley, 1995) or by ligation of lumbar dorsal
roots (Choi et al., 1996) or nerves (Steel et al., 1994). NO also
contributes to withdrawal hyperalgesia in rats made tolerant to
opioid-induced peripheral antinociception (Aley and Levine, 1997a,b).
Furthermore, the NOS inhibitor
NG-methyl-L-arginine
(L-NMA) suppresses activity in lumbar dorsal roots
originating from a sciatic neuroma (Wiesenfeld-Hallin et al., 1993).
L-NMA also reduces thermal hyperalgesia produced by chronic
constriction injury of the sciatic nerve or hindpaw inflammation (Moore
et al., 1993; Thomas et al., 1996; Lawand et al., 1997). Clearly, there
is abundant evidence that NO plays a role in nociceptive signaling.
When studying NO, it is important to evaluate cGMP, because NO
stimulates guanylyl cyclase, and many of the cellular effects of NO are
the result of NO-induced increases in the level of cGMP (Jaffrey and
Snyder, 1995). NO-donating compounds stimulate marked elevation of cGMP
levels in cultured rat DRG neurons (Dymshitz and Vasko, 1994), and
activation of NOS by the algesic agent bradykinin increases cGMP in rat
DRG neurons (Harvey and Burgess, 1996).
Because interactions between inflammatory mediators have been reported
and because there are interactions at the second messenger level, it is
important to evaluate whether NO contributes to the prototype
hyperalgesia and sensitization produced by PGE2. In this
study, therefore, we tested such a contribution by NO to hyperalgesia
produced by the inflammatory mediator PGE2, and we investigated mechanisms underlying the induction of hyperalgesia by NO,
which have not been well delineated.
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MATERIALS AND METHODS |
Animals. Experiments were performed on male Sprague
Dawley rats (200-250 gm; Bantin-Kingman, Fremont, CA). Animals were
housed in groups of two under a 12 hr light/dark cycle. Food and water were available ad libitum. All behavioral testing was done
between 10:00 A.M. and 4:00 P.M. Experiments were performed with the
approval of the Institutional Animal Care Committee of the University
of California at San Francisco.
Behavioral testing. The nociceptive flexion reflex was
quantified with a Basil analgesymeter (Stoelting, Chicago, IL), which applies a linearly increasing mechanical force to the dorsum of the
rat's hindpaw. Before the experiments, rats were exposed to the
paw-withdrawal testing procedure for 3 hr (1 hr/d for 3 d). On the
day of the experiment, rats were exposed to the same procedure for 1 hr, and the baseline threshold was determined as the mean of the six
readings before the administration of the test agent (Aley and Levine,
1997a,b). The mean ± SEM baseline threshold before treatments for
the rats used in these experiments was 108.0 ± 0.4 gm
(n = 260). Mechanical threshold was redetermined at
three time points (15, 20, and 25 min) after administration of a
hyperalgesic agent. The mean of these three readings was considered to
be the paw-withdrawal threshold because of hyperalgesic agent
administration, and this value was used to calculate the percentage
change from the baseline threshold for each paw. To determine the
timing of onset of action of the hyperalgesic agents, the mechanical
threshold was also measured at 1 min intervals for 5 min after their
administration, whereas the time course was determined by measuring the
mechanical threshold at 30-60 min intervals for 2-4 hr.
Drug administration. The following drugs used in this study
were obtained from Sigma (St. Louis, MO): PGE2,
L-arginine (L-Arg), D-arginine
(D-Arg), L-NMA,
NG-methyl-D-arginine
(D-NMA), 8-bromo-cAMP, and 3-morpholino-sydnonimine (SIN-1). Forskolin,
2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine HCl (CGS21680), and
(±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene HBr
(8-OH-DPAT) were obtained from Research Biochemicals (Natick, MA).
WIPTIDE was obtained from Peninsula Laboratories (Belmont CA),
and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and protein
kinase A catalytic subunit (PKACS) were obtained from Calbiochem (La
Jolla, CA). The selection of the drug doses used in this study was
based on dose-response curves determined during this and previous
studies (Aley and Levine, 1997a,b). The stock solution of
PGE2 (1 µg/2.5 µl) was prepared in 10% ethanol, and further dilutions were made in saline; the final concentration of
ethanol was  1%. 8-bromo-cAMP, L-NMA, D-NMA,
PKACS, and WIPTIDE were dissolved in saline. ODQ was
dissolved in DMSO and diluted with saline (final concentration of DMSO,
10%). CGS21680 and 8-OH-DPAT were dissolved in deionized water. All
drugs administered intradermally were in a volume of 2.5 µl/paw. For
test agents with low cell membrane permeability (i.e., WIPTIDE and
PKACS), 2 µl of distilled water was coinjected first, in the same
syringe as the test agent to produce hypo-osmotic shock and thus
facilitate cell permeability (Taiwo and Levine, 1989; Khasar et al.,
1995). When drug combinations were used, they were administered from
the same syringe in such a way that the drug mentioned first reached
the intradermal site first. Such combinations of agents were separated
in the syringe by a small air bubble to prevent their mixing in the
syringe. Whenever an inhibitor was included, it was injected first.
Cell culture and in vitro electrophysiology.
Primary cultures of adult rat lumbar DRG neurons were prepared as
described previously (Gold et al., 1996b). Culture medium consisted of
minimal essential medium [University of California at San Francisco
(UCSF) Cell Culture Facility] with 10% fetal bovine serum (Life
Technologies, Gaithersburg, MD) and 1000 U/ml each penicillin
and streptomycin (UCSF Cell Culture Facility). Ganglia were dissected
free, desheathed in cold culture medium, and then incubated for 2 hr at
37°C in culture medium with 0.125% collagenase. After an additional
10 min digestion in 0.25% trypsin, cells were mechanically dispersed by trituration with a fire-polished Pasteur pipette. Cells were plated onto glass coverslips coated with laminin (Life Technologies) and poly-DL-ornithine (Sigma) and were maintained in
culture medium with nerve growth factor (Life Technologies) at 37°C
under 3% CO2. Neurons were used within 24 hr of plating
before appreciable outgrowth of neurites at a time when small diameter
(20-30 µm) neuronal cell bodies express properties of nociceptors
(Gold et al., 1996b). Drugs were added via the bath, which continuously perfused the recording chamber at 1-2 ml/min. Experiments were performed at room temperature (21-24°C).
Whole-cell patch-clamp recordings were performed on small diameter
(<30 µm) neurons in 1-d-old cultures of dissociated DRGs from adult
rats, using an Axopatch 200B amplifier with pClamp6 acquisition and
stimulation programs (Axon Instruments, Foster City, CA). Data were
low-pass-filtered at 5 kHz and acquired at a sampling rate of 10 kHz.
Voltage-clamp experiments were performed with 2-5 M electrodes
filled with (in mM): CsCl 140, NaCl 10, CaCl2
0.1, MgCl2 2, EGTA 11, HEPES 10, MgATP 2, and LiATP 1, pH
adjusted to 7.2 with Tris base. Bath consisted of (in
mM): NaCl 35, tetraethylammonium chloride 30, choline chloride 65, CaCl2 0.5, MgCl2 5, HEPES
10, and glucose, pH adjusted to 7.4 with NaOH, and osmolality
adjusted to 325 mOsm with sucrose. Tetrodotoxin (50 nM) was
added to the bath. Capacitance and series resistance was compensated
(>80%), and leak subtraction was performed with a P/4 protocol. After obtaining a current-voltage relationship for TTX-R
INa, a 25 msec depolarizing test pulse was applied
every 20 sec to monitor the size of the current during the experiment.
A voltage that produced approximately half of the maximal current
activation was used for the test pulse, because the greatest increase
in TTX-R INa produced by PGE2 is seen at this
part of the current-voltage relationship. Experimental and control
neurons were studied alternately on the same day for each
comparison.
Statistical analysis. Data are presented as mean ± SEM; statistical significance was determined by ANOVA followed
by Scheffe's post hoc test; and p < 0.05 was considered statistically significant.
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RESULTS |
L-NMA, but not ODQ, blocks
PGE2 hyperalgesia
We first determined whether NO contributes to
PGE2-induced hyperalgesia and whether guanylyl cyclase is
involved (Fig. 1). Injection of
L-NMA (1 µg), a competitive NOS inhibitor, preceding PGE2 in the same syringe significantly attenuated
PGE2-induced hyperalgesia to mechanical stimuli
(p < 0.05). Injection of L-NMA (1 µg) 5 min after PGE2, when hyperalgesia is already
well established (Ouseph et al., 1995), significantly reversed
hyperalgesia. L-NMA alone had no effect on mechanical
nociceptive threshold. The inactive stereoisomer of L-NMA,
D-NMA (10 µg), was without effect on
PGE2-induced hyperalgesia. Injection of the guanylyl
cyclase inhibitor ODQ (1 µg) before PGE2 had no
effect.
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Figure 1.
Reduction of mechanical nociceptive threshold
(hyperalgesia) produced by PGE2 15 min after injection
(PGE2) (100 ng; n = 12),
L-NMA (1 µg; n = 6) plus
PGE2 (L-NMA/PGE2)
(n = 12; p < 0.05),
PGE2 5 min after injection [PGE2(5')]
(n = 6), L-NMA 5 min after
PGE2 (PGE2/L-NMA5'post)
(n = 6; p < 0.05),
D-NMA (10 µg) plus PGE2
(D-NMA/PGE2) (n = 6),
ODQ (1 µg) plus PGE2 (ODQ/PGE2)
(n = 6), and L-NMA
(n = 6) on mechanical paw-withdrawal threshold in
rats. In this and subsequent figures, *p < 0.05. Higher values indicate greater hyperalgesia. The data for
one behavioral experimental group, PGE2, is repeated
in more than one figure for ease of comparison.
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L-NMA inhibits PGE2-induced potentiation of
TTX-R Ina in vitro
PGE2 sensitizes cultured small diameter DRG neurons
and potentiates TTX-R INa (England et al., 1996; Gold et
al., 1996a). We tested whether endogenous NO derived from the primary
afferent is necessary for potentiation of TTX-R INa by
PGE2. One hundred micromolar L-NMA
(n = 15) or D-NMA (n = 14)
was added to the extracellular bath for 15 min before the application
of 1 µM PGE2. The mean PGE2-induced increase in the size of peak TTX-R
INa elicited by a depolarizing voltage step was
significantly smaller in the cells treated with L-NMA than
in those treated with D-NMA (p < 0.05) (Fig. 2).
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Figure 2.
L-NMA reduces the
PGE2-induced potentiation of TTX-R INa. TTX-R
INa was monitored by test pulses given every 20 sec.
Voltage of the test pulse ( 20 to 5 mV) was selected for each neuron
to give an approximately half-maximal current amplitude. One hundred
micromolar D-NMA or L-NMA was included in the
bath for 15 min before exposure to 1 µM PGE2.
PGE2 typically causes a potentiation of TTX-R
INa in approximately half of cells tested (Gold et al.,
1996a,b). Experiments were alternated between using the active and
inactive enantiomers of the NOS inhibitor, and data from all neurons
were used, including those in which the current was not affected by
PGE2. Peak current amplitudes were normalized to mean of
the baseline measurements.
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L-NMA blocks hyperalgesia induced by CGS21680
and 8-OH-DPAT
We tested whether NOS activity is required to produce hyperalgesia
induced by other peripherally acting hyperalgesic agents. As shown
previously (Taiwo and Levine, 1989, 1990, 1991; Taiwo et al.,
1992), the intradermal injection of CGS21680 (A2
adenosine receptor agonist; 1 µg) and 8-OH-DPAT (5HT1A
serotonergic agonist; 1 µg) produced mechanical hyperalgesia (Fig.
3). Injection of L-NMA (1 µg) with CGS21680 and 8-OH-DPAT significantly attenuated the
resulting hyperalgesia (p < 0.05) (Fig. 3),
similar to its effect on PGE2-induced hyperalgesia.
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Figure 3.
Reduction of the paw-withdrawal threshold by the
A2 adenosine agonist CGS21680 after
injection (1 µg; n = 6), L-NMA plus
CGS21680 (L-NMA/CGS) (n = 6; p < 0.05), 5HT1A agonist
8-OH-DPAT after injection (1 µg; n = 6), and L-NMA plus 8-OH-DPAT
(L-NMA/8-OH) (n = 6; p < 0.05) on mechanical paw-withdrawal
threshold in rats.
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L-NMA blocks hyperalgesia induced by 8-bromo-cAMP and
forskolin but not by PKACS
To determine whether the contribution of NO to hyperalgesia is
attributable to interaction of NO with the cAMP second messenger system
and at what level in the cAMP second messenger pathway NO is required,
we evaluated the effect of L-NMA on the hyperalgesia produced by different components of the pathway. Five minutes after the
intradermal injection of 8-bromo-cAMP (10 µg), forskolin (10 µg),
or PKACS (the catalytic subunit of PKA; 15 U), near-maximal mechanical
hyperalgesia was present. Injection of L-NMA (1 µg) before 8-bromo-cAMP and forskolin, but not PKACS, resulted in reduced
hyperalgesia (Fig. 4). Injection of
WIPTIDE inhibited PKACS hyperalgesia, as it did 8-bromo-cAMP and
forskolin hyperalgesia, which indicates that the isolated catalytic
subunit of PKA appears to produce hyperalgesia through the same
catalytic action as PKA.
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Figure 4.
Reduction of the paw-withdrawal threshold by
forskolin (10 µg) 5 min after injection (Forsk)
(n = 8), L-NMA plus forskolin
(L-NMA/Forsk) (n = 6;
p < 0.05), WIPTIDE plus forskolin
(WIPTIDE/Forsk) (n = 10;
p < 0.05), 8-bromo-cAMP after injection
(8brcAMP) (1 µg; n = 12),
L-NMA plus 8-bromo-cAMP
(L-NMA/8brcAMP) (n = 8;
p < 0.05), WIPTIDE plus 8-bromo-cAMP
(WIPTIDE/8brcAMP) (n = 6;
p < 0.05), PKACS (15 U;
n = 12), WIPTIDE plus PKACS
(WIPTIDE/PKACS) (n = 6;
p < 0.05), and L-NMA plus PKACS
(L-NMA/PKACS) (n = 6;
not statistically significant) on mechanical paw-withdrawal threshold
in rats.
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Administration of NO donor or precursor induces hyperalgesia
As shown in Figure 5, A
and D, intradermal injection of the NOS substrate
L-Arg (10 ng to 10 µg) or the NO donor SIN-1 (10 ng to 40 µg) caused a dose-dependent decrease in the paw-withdrawal threshold.
This hyperalgesia was inhibited by the guanylyl cyclase inhibitor ODQ
but was unaffected by the PKA inhibitor WIPTIDE (Fig.
6A). This hyperalgesic
action of NO contrasts with the guanylyl cyclase-independent mechanism
by which NO facilitates PGE2-induced hyperalgesia (Fig.
6B). In control experiments, D-Arg did
not induce hyperalgesia (Fig. 6A).
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Figure 5.
A, Dose-response curve for
L-Arg-induced hyperalgesia (n = 6).
B, Latency to onset of L-Arg-induced (10 µg) mechanical hyperalgesia (n = 6).
C, Time course of L-Arg-induced mechanical
hyperalgesia (n = 6). D,
Dose-response curve of SIN-1-induced hyperalgesia
(n = 6). E, Latency to onset of
SIN-1-induced hyperalgesia (10 µg; n = 6).
F, Time course of SIN-1-induced hyperalgesia
(n = 8).
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Figure 6.
A, Change in mechanical paw
withdrawal after injection of L-Arg
(L-Arg) (10 µg; n = 12), ODQ plus L-Arg (ODQ/L-Arg)
(n = 6), WIPTIDE plus L-Arg
(WIPTIDE/L-Arg) (n = 6),
L-NMA plus L-Arg
(L-NMA/L-Arg)
(n = 6), D-NMA/L-Arg
(n = 6), D-Arg
(D-Arg) (10 µg; n = 6), PGE2 (PGE2) (n = 12), WIPTIDE plus PGE2 (WIPTIDE/PGE2)
(n = 12), ODQ plus PGE2
(ODQ/PGE2) (n = 8),
SIN-1 (10 µg; n = 6), ODQ plus
SIN-1 (ODQ/SIN) (n = 8), and
WIPTIDE plus SIN-1 (WIPTIDE/SIN)
(n = 6). B, Change in mechanical
paw-withdrawal threshold after injection of PGE2
(n = 12), L-NMA plus PGE2
(L-NMA/PGE2) (n = 6),
SIN-1 (SIN) (100 ng; n = 6),
and L-NMA plus ODQ plus PGE2 plus SIN-1
(L-NMA/ODQ/PGE2/SIN) (100 ng;
n = 12).
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Exogenous sources of NO can reconstitute the facilitatory effect of
endogenous NO on PGE2-induced hyperalgesia
We tested whether NO from exogenous sources can mimic the ability
of endogenous NO to facilitate PGE2-induced hyperalgesia. To inhibit endogenous NO production, L-NMA was coinjected
into the paw with PGE2, which reduced the
PGE2-induced decrease in paw-withdrawal threshold by
~62% (Fig. 1). As shown in Figure 6B, coinjection
of the NO donor compound SIN-1 with L-NMA and PGE2 restored the PGE2-induced decrease in
paw-withdrawal threshold to a value similar to that observed when
endogenous NOS was not inhibited (i.e., in the absence of
L-NMA). The 100 ng dose of SIN-1 that facilitated
PGE2-induced hyperalgesia was insufficient to induce
hyperalgesia by itself (Fig. 5D). To further preclude the
possibility that NO-induced (cGMP-dependent) hyperalgesia played a role
in this reconstitution experiment, the guanylyl cyclase inhibitor ODQ
was coinjected with PGE2, L-NMA, and
SIN-1.
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DISCUSSION |
In this study, we found that NO, but not cGMP, contributes to
initiation and maintenance of hyperalgesia and sensitization produced
by the inflammatory mediator PGE2. We also found that the
independent hyperalgesia produced by NO depends on cGMP and may require
a higher concentration than that for facilitation of PGE2
hyperalgesia and sensitization. The reduction in
PGE2-induced potentiation of TTX-R INa in
L-NMA treated cells parallels the effect of
L-NMA on PGE2-induced hyperalgesia, suggesting
that both phenomena depend on NO for full expression and that cells other than the one being recorded from are not required for these effects of PGE2 or NO. Although many effects of NO have
been reported to be mediated by guanylyl cyclase activity (Jaffrey and
Snyder, 1995), we observed that the guanylyl cyclase inhibitor ODQ had no effect on PGE2-induced hyperalgesia, suggesting that
facilitation of PGE2-induced hyperalgesia by NO and the
other mechanisms of PGE2-induced hyperalgesia do not
involve NO signaling through guanylyl cyclase.
Additional experiments attempted to determine where NO might act to
facilitate PGE2 hyperalgesia. Evidence suggests that
initiation of PGE2-induced hyperalgesia is attributable to
action of PGE2 at an E-type prostaglandin receptor on the
primary afferent nociceptor terminal, activation of a stimulatory
G-protein which then activates adenylyl cyclase, followed by an
increase in the level of cAMP and activation of PKA (Taiwo and Levine,
1989, 1991; Khasar et al., 1995). We observed that hyperalgesia
produced by 8-bromo-cAMP (which activates PKA) and forskolin (which
activates adenylyl cyclase) was attenuated by L-NMA in a
manner similar to the effect of L-NMA on PGE2
hyperalgesia, but that in contrast, hyperalgesia produced by injection
of PKACS (to mimic activity of endogenous PKA) was not affected by
L-NMA. These results suggest that NO might be required for
activation of PKA after administration of PGE2. Of note,
others have shown that NO can modulate activity of kinases
(Gopalakrishna et al., 1993; Burgstahler and Nathanson, 1995; Studer et
al., 1996; Minamino et al., 1997). The fact that L-NMA
inhibited hyperalgesia induced by two other direct-acting hyperalgesic
agents (CGS21680, an A2 adenosine receptor agonist, and
8-OH-DPAT, a 5HT1A serotonin receptor agonist) suggests
that dependence on NO is a general feature of hyperalgesia induced via
the cAMP second messenger system.
Additional experiments revealed that there was an independent
NO-induced hyperalgesia that depended on the cGMP second messenger pathway and not on the cAMP pathway (Fig. 6B). We
hypothesize that a relatively low level of NO can facilitate
cAMP-dependent hyperalgesia induced by PGE2 and other
inflammatory mediators, whereas stimulated increases in NO to higher
levels might be required to induce the cGMP-dependent hyperalgesia.
Supporting this hypothesis, when endogenous NO production was blocked,
the level of NO donor required to facilitate PGE2-induced
hyperalgesia was at least an order of magnitude lower than the level
required for NO to induce hyperalgesia by itself. However, it is not
possible to know whether exogenous NO has ready access to the necessary
sites. Also, we cannot determine from our data whether basal levels of constitutively synthesized NO are sufficient to facilitate
PGE2-induced hyperalgesia or whether PGE2
stimulates synthesis of NO (to a level lower than that required to
induce cGMP-dependent hyperalgesia). However, it appears that
constitutive synthesis of NO does not affect baseline nociceptive
threshold, because inhibition of NOS does not alter paw-withdrawal
threshold. Finally, although our in vitro patch-clamp data
imply that the NO that facilitates PGE2-induced hyperalgesia is synthesized in DRG neurons and acts on DRG neurons, our
data do not determine the site of action for cGMP-dependent NO-induced
hyperalgesia. Clearly, further study is needed to determine concentration and site requirements for the two distinct effects of NO
that we have evaluated.
NO is generated in significant concentrations at sites of inflammation
in which multiple hyperalgesic inflammatory mediators, such as
PGE2, adenosine, or serotonin, are also produced.
Although the evidence for a role of NO in clinical pain is limited, NO may facilitate the hyperalgesia induced by those mediators using the
cAMP second messenger pathway and may also have an independent cGMP-dependent hyperalgesic effect. If both of these contributions are
clinically significant, different therapies for these two distinct
mechanisms of NO may be needed for successful pharmacological treatment
of inflammatory pain.
| |
FOOTNOTES |
Received April 23, 1998; revised June 12, 1998; accepted June 17, 1998.
This work was supported by National Institutes of Health Grant NS21647.
We thank Dr. David Bredt for many discussions about NO/cGMP signaling
and Drs. David Reichling and Kimberly Tanner for their careful scrutiny
of this manuscript.
Correspondence should be addressed to Dr. Jon D. Levine, National
Institutes of Health Pain Center (UCSF), C-522 Box 0452, University of
California at San Francisco, San Francisco, CA 94143-0452.
| |
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Top
Abstract
Introduction
