Department of Anatomy and Physiology, Meharry Medical College,
Nashville, Tennessee 37208
 |
INTRODUCTION |
Endomorphin-1
(Tyr-Pro-Trp-Phe-NH2) and endomorphin-2
(Tyr-Pro-Phe-Phe-NH2) isolated recently from the
bovine (Zadina et al. 1997
) and human (Hackler et
al. 1997) brains have been suggested to be the endogenous
ligands for the µ-opioid receptor (Zadina et al.
1997). The presence of a dense aggregation of endomorphin-like immunoreactive elements in the superficial dorsal horns of the medulla
and spinal cord indicates that endomorphins are likely to modulate
nociceptive transmission (Martin-Schild et al. 1997, 1999; Wu et al. 1999). Behavioral evidence
indicates that the peptides produce a potent and prolonged analgesia in
mice (Zadina et al. 1997) and rats (Przewlocka et
al. 1999).
We have been investigating the opioid-mediated modulation of
somatosensory mechanisms in the medullary dorsal horn (trigeminal nucleus caudalis) (Mokha 1992; Wang et al. 1996,
1999; Zhang et al. 1996), a region considered
important for the relay of somatosensory information originating from
nociceptors, thermoreceptors, and mechanoreceptors in the orofacial
region (reviewed in Light 1992; Sessle
1987). Glutamate, a putative excitatory neurotransmitter, is
present in trigeminal primary afferent fibers (Clements et al.
1991; Watkins and Evans 1981; Wilcox
1993), and acts on N-methyl-D-aspartic acid (NMDA), non-NMDA ionotropic and metabotropic receptors. NMDA, non-NMDA ionotropic and metabotropic receptors are present in the
medullary dorsal horn, especially in its superficial laminae (Kondo et al. 1995; Tallaksen-Greene et al.
1992). The NMDA receptor, in particular, has been shown
repeatedly to be involved in mediating nociceptive neurotransmission
and neural plasticity (hyperalgesia) in the spinal dorsal horn
(reviewed in Wilcox 1993; Willis et al.
1996).
The functional significance of endomorphins in the trigeminal system
remains unknown. Further, the role that endomorphins might play in
modulating the NMDA-evoked and natural stimulus-evoked responses of
nociceptive neurons has not been investigated previously in in vivo
studies. The present study was therefore designed to investigate the
effects of endomorphins administered microiontophoretically on the
NMDA-evoked and natural cutaneous stimulus-evoked responses of
physiologically characterized nociceptive neurons in the medullary dorsal horn.
| |
METHODS |
Subjects, recording, and drug administration procedures
Techniques used for animal preparation, neuronal recording, and
classification of trigeminal neurons have been described previously (Wang et al. 1996, 1999). Experiments were performed on
34 male Sprague-Dawley rats (body wt 240-350 g, Harlan Sprague Dawley, Indianapolis, IN) anesthetized with urethan (1.5 g/kg ip, initial dose). Subsequently, a smaller dose of urethan was given intravenously, if and when necessary, to maintain a stable level of anesthesia. The
electrical activity of the heart and rectal core temperature were
monitored continuously. The exposed surface of the medulla was covered
with agar (4% agar in normal saline at ~40°C) to improve the
stability of recording from neurons in the superficial dorsal horn of
the medulla. Extracellular single-unit recordings were made from
nociceptive neurons using the central barrel of a seven-barrel micropipette (MS-7PB, tip diam 5-8 µm, impedance: 4-6 M
, Medical Systems, Harvard apparatus). The remaining barrels were filled with
freshly made solutions of the following drugs: endomorphin-1 (10 mM in
double-distilled water, pH 5.0); endomorphin-2 (10 mM in
double-distilled water, pH 5.0);
[D-Ala2,
N-Me-Phe4,
Gly5-ol]-enkephalin (DAMGO, 10 mM in
double-distilled water, pH 4.0); NMDA (50 mM in 150 mM NaCl, pH 8.0);
-aminobutyric acid (GABA, 250 mM in 160 mM NaCl, pH 4.5); naloxone
hydrochloride (10 mM in double-distilled water, pH 5.0); and 2 M NaCl
for current balancing. All drugs, except NMDA, were ejected with
positive current, whereas NMDA was ejected with negative current.
Retaining currents of 5-10 nA were used routinely to prevent drug
diffusion. Controls (pH and current) were performed as described
previously (Zhang et al. 1996). All drugs, except
endomorphin-1 and endomorphin-2, were obtained from Sigma Chemical (St
Louis, MO). Endomorphin-1 and endomorphin-2 were purchased from
Research Biochemicals International (Natick, MA).
Stimulation, data analyses, and histological procedures
Neurons were characterized as nociceptive specific (NS,
responding only to noxious stimuli) and wide dynamic range (WDR,
responding to noxious and nonnoxious stimuli). Noxious mechanical
stimuli were applied briefly (3-5 s) at 3- to 5-min intervals with an arterial clip. Noxious thermal stimuli (<58°C) were applied for 15 s starting from a baseline of 36°C (rise time:10 s; 2°C/s) at 3- to 5-min intervals using a radiant heat stimulator (Beck et al. 1974). Brush stimuli were applied at 3- to 5-min
intervals using a hand-held camel hair brush. Responses (total number
of spikes/stimulus) evoked by at least three to five applications of
natural stimuli or responses evoked by cyclical administration of NMDA
over 5 min prior to the application of a test drug served as the
control responses. In most cases, iontophoretic currents for NMDA were
adjusted to generate peak firing rates of 40-60 Hz. Responses during
iontophoresis of a test drug were compared with the control responses.
The effect produced by the application of a test drug was defined as
inhibitory or excitatory only when the NMDA-evoked responses differed
from the mean of the control responses by ±2 SD in the same direction
(facilitation or inhibition). The peak percentage change in the number
of spikes was calculated by comparing the mean of the lowest number of
spikes evoked by three to five applications of NMDA following
applications of a test drug to the control responses. Group data are
expressed as means ± SE. Statistical analysis was performed using
the paired t-test and one-way ANOVA (followed by
Student-Newman-Keuls test), and a probability level of <0.05 was
considered significant. Some recording sites were marked and
reconstructed as described previously (Wang et al. 1996,
1999).
| |
RESULTS |
Effects of microiontophoretically applied endomorphin-1 and
endomorphin-2 were respectively tested on the NMDA-evoked responses of
37 neurons (8 NS, 29 WDR) and 35 neurons (4 NS, 31 WDR) in the
medullary dorsal horn. Endomorphin-1 (10-70 nA) produced a peak
inhibitory effect of 54.1 ± 2.96% (n = 34, P < 0.001) on the NMDA-evoked responses in 92%
(34/37) of neurons (8 NS, 26 WDR, Fig.
1B). In general, the
inhibitory effect was short-lasting (Fig. 1B). Facilitation
was observed in three neurons (3 WDR). Similarly, endomorphin-2 (10-70
nA) produced a peak inhibitory effect of 63.6 ± 3.61%
(n = 32, P < 0.001) on the NMDA-evoked responses in 91% (32/35) of neurons (4 NS, 28 WDR, Fig.
1A). The inhibitory effect was also short-lasting (Fig.
1A). Facilitation was observed in three neurons (3 WDR).
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
A: endomorphin-2 and [D-Ala2,
N-Me-Phe4, Gly5-ol]-enkephalin
(DAMGO) significantly reduced the
N-methyl-D-aspartate (NMDA)-evoked responses
[counts = spikes/bin (1 s) ] of a wide dynamic range (WDR)
neuron in the deeper dorsal horn of the medulla and naloxone
antagonized the inhibitory effects of endomorphin-2 and DAMGO.
B: endomorphin-1 significantly reduced the NMDA-evoked
responses and naloxone antagonized the inhibitory effect of
endomorphin-1 on the same neuron. Note the different scale range used
for the x axis in A and
B.
|
|
Microiontophoretic applications of endomorphin-1 and endomorphin-2 were
tested respectively on the natural stimulus-evoked responses in 16 neurons (7 NS, 9 WDR) and 17 neurons (5 NS, 12 WDR). Endomorphins
primarily reduced the natural-stimulus-evoked responses of nociceptive
neurons. The inhibitory effect on a nociceptive-specific neuron located
in the superficial dorsal horn is illustrated in Fig.
2. The inhibitory effects of endomorphins
were more prolonged on noxious stimulus-evoked responses than
NMDA-evoked responses, particularly the inhibition evoked by
endomorphin-2 (Fig. 2, A and B). Endomorphin-1
produced a peak inhibitory effect of 55.6 ± 6.87%
(n = 9, 2 NS, 7 WDR, Fig.
3A) and 74.8 ± 7.80%
(n = 6, 4 NS, 2 WDR, Fig. 3A) on the pinch-
and heat-evoked responses, respectively. In contrast, endomorphin-1
produced only a small reduction of 11.3 ± 6.01%
(n = 6, 6 WDR, Fig. 3A) on the brush-evoked responses. Similarly, endomorphin-2 produced a peak inhibitory effect
of 57.8 ± 5.7% (n = 10, 2 NS, 10 WDR, Fig.
3B) and 86.8 ± 6.61% (n = 4, 3 NS, 1 WDR, Fig. 3B) on the pinch- and heat-evoked responses,
respectively. The brush-evoked responses were reduced only 7.1 ± 6.6% (n = 10, 10 WDR, Fig. 3B).
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Inhibitory effects of endomorphin-1 and endomorphin-2 on the
heat-evoked responses of a nociceptive specific neuron in the
superficial dorsal horn of the medulla (inset,
 ). Naloxone reduced the inhibitory effect of endomorphin-2.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
A: the inhibitory effects of endomorphin-1 on the
natural stimulus-evoked responses of neurons in the superficial and
deeper dorsal horn of the medulla. Each column represents the mean ± SE. *, significant (P < 0.05) differences
between the inhibitory effects on the noxious vs. the nonnoxious
(brush) stimulus-evoked responses (ANOVA followed by
Student-Newman-Keuls test). B: the inhibitory effects of
endomorphin-2 on the natural stimulus-evoked responses of neurons in
the superficial and deeper dorsal horn of the medulla. Each column
represents the mean ± SE. #, significant difference between the
inhibitory effects on the heat- vs. the pinch-evoked responses
(P < 0.05, ANOVA followed by Student-Newman-Keuls
test).
|
|
Naloxone applied iontophoretically at currents that blocked the effects
of DAMGO, a µ-opioid receptor agonist, blocked or reduced the
inhibitory effects of endomorphin-1 and endomorphin-2 by 70.39%
(n = 9, P < 0.01, Fig. 1A)
and 65.96% (n = 10, P < 0.01, Fig.
1B) on the NMDA-evoked responses, respectively. The
antinociceptive effects of endomorphins on the noxious stimulus-evoked
responses were also reduced by iontophoretic application of naloxone
(Fig. 2). However naloxone applied at identical parameters
(current × time) did not alter the inhibitory effects of GABA on
the same neurons.
| |
DISCUSSION |
This is the first in vivo electrophysiological study that examined
the effects of endomorphins on the NMDA-evoked and
natural-stimulus-evoked responses of nociceptive neurons. The present
results indicate that endomorphins primarily produced inhibitory
effects on the NMDA-evoked responses and the noxious
natural-stimulus-evoked responses through a naloxone-sensitive opioid
receptor in the medullary dorsal horn. Our observations are consistent
with the behavioral observations demonstrating an analgesic effect of
endomorphins administered intracerebroventricularly (Zadina et
al. 1997) or intrathecally in mice (Stone et al.
1997; Zadina et al. 1997) and rats
(Przewlocka et al. 1999), and with the
electrophysiological observations demonstrating the inhibitory effects
of endomorphins on the C-fiber-evoked responses of spinal dorsal horn
neurons in the rat (Chapman et al. 1997). The inhibitory
effect of endomorphins is presumably mediated by postsynaptic
inhibitory mechanisms since endomorphins have been shown to
hyperpolarize substantia gelatinosa neurons (Wu et al.
1999). However, presynaptic mechanisms may also play a role
since endomorphins have been shown to decrease peripheral
stimulus-evoked excitatory postsynaptic potentials (Wu et al.
1999) and inhibit high-threshold Ca2+
channel currents (Higashida et al. 1998).
The time course of the inhibitory effect of endomorphins on the
NMDA-evoked responses in the present study is shorter as compared with
the long-lasting effects observed in some behavioral (more than an
hour) (Przewlocka et al. 1999; Zadina et al.
1997) and electrophysiological (Chapman et al.
1997) studies. However, endomorphins did produce a
longer-lasting inhibitory effect on the noxious stimulus-evoked
responses in the present study. These differences in the duration of
the inhibitory effect could result from a number of factors including,
different intracellular effector mechanisms or different mechanisms of
action at a membrane level versus at an intracellular level and
different firing patterns generated by NMDA and noxious stimuli.
Further, endomorphin-2 produced a longer-lasting effect compared
with that of endomorphin-1. It is possible that both shared and
separate intracellular effector mechanisms mediate effects of
endomorphins. Although many subunits of the Gi
protein, such as Gi1, Gi3
and GZ, are involved in mediating the
antinociceptive effects of both endomorphin-1 and endomorphin-2, Gi2 is only involved in mediating the
antinociceptive effect of endomorphin-2
(Sánchez-Blázquez et al. 1999). Further,
endomorphin-2 appears to be more prevalent in the superficial laminae
of the medullary dorsal horn than endomorphin-1 (Martin-Schild
et al. 1997, 1999; Wu et al. 1999). In contrast
to our findings, behavioral observations indicate that the
antinociceptive effect of endomorphin-2 is less potent than
endomorphin-1 administered intrathecally (Przewlocka et al.
1999). The differences may arise from differential modulation of sensory versus motor circuits and different drug concentrations attained in behavioral and electrophysiological studies. Our
observations that the inhibitory effect of endomorphins is
modality-specific are consistent with the endomorphin-2 induced
selective modulation of C-fiber-evoked responses reported in the spinal
dorsal horn (Chapman et al. 1997).
Naloxone applied at a current that antagonized the effects of DAMGO, a
selective µ-opioid receptor agonist, reduced the inhibitory effects
of endomorphins, indicating that the inhibitory effect induced by
endomorphins is mediated by µ-opioid receptor activation. Therefore
the predominantly inhibitory modulation of the NMDA-evoked and
noxious-stimulus-evoked responses of nociceptive neurons suggests that
endomorphins are involved in producing an antinociceptive effect at the
level of the medullary dorsal horn by acting at the µ-opioid receptor.
We are thankful to C. A. Fairbanks (from the laboratory of Dr.
George L. Wilcox at the University of Minnesota) for the generous gift
of endomorphins used in the initial experiments.
This work was supported by National Institutes of Health Grants
DE-10903, RR-3032, DERR-10595, and GM-08037. C. A. Flores was supported
by National Institute of Mental Health Training Grant T32 MH-19843.
Address for reprint requests: S. S. Mokha, Dept. of Anatomy and
Physiology, Meharry Medical College, 1005 D.B. Todd Blvd., Nashville,
TN 37208.
The costs of publication of this article were defrayed in part
by the payment of page charges. The article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
TOP
ABSTRACT
INTRODUCTION
