© 1995 American Heart Association, Inc.
Articles |
Attenuation of the Exercise Pressor Reflex
Effect of Opioid Agonist on Substance P Release in L-7 Dorsal Horn of Cats
From the Harry S. Moss Heart Center, Departments of Internal Medicine and Physiology, University of Texas Southwestern Medical Center at Dallas.
Correspondence to Dr L. Britt Wilson, Harry S. Moss Heart Center, Department of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Using
-chloralose–anesthetized cats, we
studied blood pressure and heart rate responses to static contraction
and passive stretch of the triceps surae muscle before and after
microdialyzing the µ-opioid agonist
[D-Ala2]-methionine enkephalinamide
(DAME, 200 µmol/L) into the L-7 dorsal horn of the spinal cord. In
addition, we measured contraction-induced substance P release in the
dorsal horn before and after drug delivery. After 92±3 minutes of
dialyzing the opioid agonist, contraction-induced increases in mean
arterial pressure and heart rate were attenuated from
control values of 58±7 mm Hg and 17±3 beats per minute to postdrug
values of 27±7 mm Hg and 10±2 beats per minute, respectively. A
similar attenuation was observed for the passive muscle stretches after
97±5 minutes of dialysis (control, 38±4 mm Hg and 8±2 beats per
minute; after drug, 23±4 mm Hg and 5±1 beats per minute). Prior
microdialysis of naloxone (300 µmol/L), a µ-antagonist,
blocked this effect, suggesting that the opioid agonist has a specific
receptor action. Naloxone alone had no effect on the pressor or
tachycardiac responses. The contraction-induced increase in
substance P–like immunoreactivity was reduced from a control value of
0.119±0.024 to 0.047±0.010 fmol/100 µL by DAME. Time-control
experiments revealed no decrease in the release of substance P–like
immunoreactivity. Thus, activation of opioid receptors modulates the
transmission of group III and IV muscle afferent nerve activity through
the L-7 dorsal horn. Presynaptic inhibition of substance P release from
muscle afferents is a potential mechanism of action for DAME.
Key Words: muscle contraction • presynaptic inhibitory effect • primary afferents • substance P
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In anesthetized cats, static contraction of the triceps surae muscle causes a reflex increase in MAP, HR, and ventilation.1 2 3 This response, referred to as the "exercise pressor reflex," is mediated via group III and IV muscle afferent nerve fibers,2 3 the majority of which synapse in the lumbar dorsal horn of the spinal cord.4 5
The exercise pressor reflex is attenuated by the intrathecal administration of an SP antagonist6 or by microinjecting the antagonist into the L-7 dorsal horn.7 In addition, SP increases in the L-7 dorsal horn region in response to static muscle contraction,8 the increase being dependent on the tension developed by the muscle.9 Therefore, SP serves as a neurotransmitter or neuromodulator for the afferent limb of the exercise pressor reflex.
Although opioid receptor agonists have been shown to attenuate the cardiovascular responses to static muscle contraction,10 the mechanism of action is unclear. Since opioid receptors have been localized to primary afferent fibers11 and various opioid agonists have been shown to reduce SP release from primary afferent neurons in vitro12 13 and in vivo,14 15 one can propose a presynaptic mechanism. Thus, attenuation of a contraction-induced increase in AP with a simultaneous reduction in SP release after the administration of the opioid agonist DAME into the L-7 dorsal horn would be evidence in support of a presynaptic mechanism of action.
Therefore, the aims of the present study were to attenuate the contraction-induced pressor response by microdialyzing DAME into the L-7 dorsal horn and to measure the contraction-induced SP release both before and after microdialyzing DAME.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Twenty-six young adult mongrel cats (2.8±0.5 kg) of either sex were used for the present study. Protocols were approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center.
Surgical Preparation
Anesthesia was induced by using a halothane
(5%)/nitrous oxide (1 to 3 L/min)/oxygen (1 to 3 L/min) mixture, and
the halothane was reduced to 2% to 3% while catheterizing a jugular
vein and a carotid artery and inserting a tracheal tube via a
tracheotomy. The inhalation anesthesia was then substituted
by intravenous -chloralose (initial dose, 60 to 80
mg/kg; maintenance, 5 to 10 mg/kg and/or sodium pentobarbital
at 1 to 3 mg/kg as needed). Supplemental doses of
anesthesia were given if the corneal and/or pupillary
reflexes were present or if a gradual increase in baseline MAP
and/or HR occurred. Blood gases and the acid-base balance were
monitored throughout the experiment (model ABL-3 blood gas
analyzer, Radiometer) and maintained within normal limits (pH
7.35 to 7.40; PCO2, 35 to 40 mm Hg;
PO2, >80 mm Hg; and plasma
HCO3-, 19 to 24 mmol/L) by mechanical
ventilation (model 661 respirator, Harvard Apparatus);
supplemental oxygen or intravenous sodium bicarbonate (1
mol/L) was administered. Body temperature was monitored via a rectal
temperature probe and maintained between 37°C and 38°C with a
heating pad and an infrared heat lamp.
A laminectomy was performed exposing the lumbosacral spinal cord. All spinal roots (dorsal and ventral) from L-5 to S-2 were unilaterally severed close to the spinal cord, maintaining the L-7 dorsal root intact. The remaining afferent nerve activity mediating the pressor reflex from the triceps surae muscle thus passed through the L-7 dorsal root.16 The peripheral cut ends of the L-7 and S-1 ventral roots were subsequently placed on bipolar platinum electrodes for stimulation. The cat was placed in a spinal unit (David Kopf Instruments) to stabilize the spine. The hind limb containing the triceps surae muscle to be contracted or stretched (ipsilateral to the cut spinal roots) was immobilized by tying the patellar tendon to a steel post. The triceps surae muscle was isolated, the calcaneus was severed, and the Achilles' tendon was attached to a force transducer (model FT10, Grass Instruments Co). By use of a Kopf stereotaxic guide (David Kopf Instruments), a CMA-10 microdialysis probe (3-mm-long membrane, 640-µm diameter, Bioanalytical Systems Inc) was inserted into the dorsal horn at the midpoint of the uncut L-7 dorsal root and perpendicular to the surface of the spinal cord.
The arterial catheter was connected to a Statham pressure transducer (model P23ID) for recording AP. MAP was derived by integrating the AP signal with a time constant of 4 seconds. HR was derived from the AP signal by using a biotachometer (Gould Instruments). Tensions that developed during the contractions and stretches were measured by the force transducer attached to the Achilles' tendon.
After the surgery, the preparation was allowed to stabilize for 2 hours before beginning an experiment, during which the probe was continually perfused at a rate of 5 µL/min with an artificial ECF containing 0.2% bovine serum albumin, 0.1% bacitracin, and the following ions (mmol/L): K+ 6.2, Cl- 134, Ca2+ 2.4, Na+ 150, P- 1.3, HCO3- 13, and Mg2+ 1.3. For all the protocols, control data were collected while this artificial ECF was being dialyzed.
Dose-Response Protocol
After a minimum of two repeatable control contractions, doses of
20 µmol/L, 200 µmol/L, and 2 mmol/L of the µ-opioid receptor
agonist DAME17 were microdialyzed sequentially into the
L-7 dorsal horn of three cats. Each dose was microdialyzed for 
40
minutes, at which time the triceps surae muscle was contracted while
recording MAP, HR, and tension. The contractions were induced
by stimulating the peripheral cut ends of the L-7 and S-1
ventral roots for 1 minute at three times MT, 40 Hz, and 0.1- to
0.3-millisecond duration.
Experimental Protocol I
Based on the above dose response, this protocol was designed to
determine the effect of 200 µmol/L DAME, microdialyzed into the L-7
dorsal horn, on the pressor and HR responses to static contraction and
passive stretch of the triceps surae muscle. Ten cats were used. Before
the administration of DAME, at least two reproducible control static
muscle contractions and stretches were performed while
recording the MAP and HR responses and developed tension as
described above. The contractions were induced by stimulating the
peripheral cut ends of the L-7 and S-1 ventral roots for 1
minute at three times MT, 40 Hz, and 0.1- to 0.3-millisecond duration.
Muscle stretch was performed by manually displacing the force
transducer until the desired tension was reached. The tensions during
the stretches were matched with the tensions developed during the
contractions.
DAME (200 µmol/L) was then microdialyzed for 40 minutes, and
either a contraction or a stretch was performed in a counterbalanced
design. Fifteen minutes later, either a stretch or a contraction
followed, respectively. Similarly, a second contraction and stretch was
performed after the DAME had been dialyzed for a total of 80
minutes.
To functionally verify probe placement, a 20% lidocaine solution was dialyzed for 40 minutes; dialysis was followed by a contraction and a stretch. The data were excluded from the study if the pressor response to contraction following lidocaine administration was not attenuated by at least 70% relative to control.
Experimental Protocol II
This protocol was designed to determine if the effect produced
by 200 µmol/L DAME microdialyzed into the L-7 dorsal horn is a
consequence of µ-opioid receptor activation or is a nonspecific
effect. Naloxone (300 µmol/L), a µ-opioid receptor
antagonist, was used to antagonize the effects of DAME.
Data from two reproducible control static muscle contractions and
passive stretches were collected from three cats. The contractions and
stretches were elicited in the same manner as described in protocol I.
Next, a 300 µmol/L naloxone solution was microdialyzed into the L-7
dorsal horn for 60 minutes. A contraction and a stretch were then
performed, with data being recorded as described previously. DAME
(200 µmol/L) was then microdialyzed for 80 minutes, at which stage
either a contraction or a stretch was again performed. A 20% lidocaine
solution was then microdialyzed as described previously.
Experimental Protocol III
The aim of this protocol was to determine if microdialysis of
200 µmol/L DAME into the L-7 dorsal horn reduced the
contraction-induced release of SP. The same surgical preparation as
described above was performed on a separate group of 10 cats. Eight
cats formed the experimental group in which 200 µmol/L DAME was
microdialyzed into the L-7 dorsal horn, and two cats served as time
controls in which artificial ECF was microdialyzed instead of the
DAME.
One-Probe Experiments
Immediately after insertion of the dialysis probes, six
sequential 100 µL dialysate samples (5 µL/min for 20 minutes each)
were collected during a 2-hour stabilization period. The sixth sample
was regarded as the control baseline SP-LI. A 5-minute static
contraction was then induced by alternately stimulating the L-7 and S-1
ventral roots (three times MT, 30 Hz, and 0.1- to 0.3-millisecond
duration). The control contraction dialysate sample was collected for
10 minutes at 5 µL/min (50 µL sample volume) with the collection
starting 25 seconds into the contraction to account for the dead space
of the dialysis probe and tubing. Two 20-minute recovery samples were
then collected. The 5-minute contraction and 10-minute dialysate
collection were then repeated, and the two contraction dialysate
samples were combined to yield a single 100 µL sample for subsequent
determination of the control contraction–induced change in SP-LI. When
one probe is used, muscle tension cannot be maintained for an adequate
time in a single contraction to allow for sufficient dialysate
collection. Two 20-minute recovery samples were then collected, after
which 200 µmol/L DAME was microdialyzed for 100 minutes (five
20-minute collections). This period of DAME dialysis was similar to
that used in protocol I. The fifth collection, after starting the DAME
dialysis, served as the precontraction baseline for SP-LI. As for the
control condition, two 5-minute contractions with the accompanying
10-minute collections followed. The contractions were separated by 40
minutes (two 20-minute collections). The dialysates from the third and
fourth contractions were combined to yield one 100 µL sample for
SP-LI analysis.
For the two cats serving as time controls, the same protocol was followed, except artificial ECF was dialyzed throughout.
Two-Probe Experiments
Two probes allowed us to collect appropriate volumes of
dialysate over half the time. For probe placement, the L-7 dorsal horn
was visualized in quarters rostrocaudally. One probe was placed between
the first and second quarters, and the other probe was placed between
the third and fourth quarters. Six 20-minute dialysate collections were
performed as described above; the dialysate from both probes was
collected into the same vial, giving a final volume of 200 µL (two
probes, 5 µL/min for 20 minutes). The dialysate (100 µL) was
pipetted into a separate vial for storage. The sixth sample served as
the baseline for the control contraction. As described above
("One-Probe Experiments"), a 5-minute contraction was induced,
during which time dialysate was collected from both probes into the
same vial; the final volume was 100 µL. Five 20-minute recovery
samples followed, after which 200 µmol/L DAME was microdialyzed for
50 minutes (five 10-minute collections [n=1]) or 100 minutes (five
20-minute collections [n=2]). The fifth recovery sample was
considered the baseline for the subsequent contraction. Thus, for the
two-probe experiments, only one control contraction and one contraction
following the dialysis of DAME were necessary to yield a 100 µL
dialysate sample. All the dialysate samples were stored at -80°C for
subsequent analysis using RIA.
RIA Procedure
This procedure has been previously described in
detail.8 Briefly, removable microplate wells (Removawell
Immulon 4, Dynatech) were coated with 1 µg purified protein A
(purified recombinant protein A, binding 1.3 mg rabbit IgG per
milligram; Pierce) in 100 µL of 0.2 mol/L sodium borate (pH 9.0).
After a 24-hour incubation period at 4°C, wells were washed three
times with an RIA buffer (0.05 mol/L sodium phosphate buffer, pH 7.4).
Fifty microliters of the purified SP antiserum, diluted in RIA buffer,
was added. The wells were then washed three times with the RIA buffer
after 8 hours of incubation at 4°C. Remaining protein binding sites
on the polystyrene-like wells were saturated by incubating with 3%
bovine serum albumin in PBS overnight at 4°C. Again, after
washing three times with the RIA buffer, standards in triplicate and
samples (100 µL) were added to the wells and incubated for 24 hours
at 4°C. [125I-Tyr8]SP-(1-11) (100 µL) was
added to the standards and the samples. These were incubated for 24
hours at 4°C. Wells were then washed three times with RIA buffer and
separated, and the radioactivity was counted for 10 minutes in a
four-well gamma counter by using a cubic spline algorithm for data
processing.
The cross-reactivity at IC50 was 100% for SP-(1-11) and SP-(2-11), 98% for [125I-Tyr8]SP-(1-11), 95% for SP-(1-9), 85% for SP-(1-7), 75% for SP-(3-11), 60% for SP-(1-6), 45% for SP-(1-4), 12% for SP-(4-11) and SP-(5-11), 5% for SP-(6-11), and <0.01% for SP-(7-11), SP-(8-11), SP-(9-11), neurokinin A, neurokinin B, somatostatin, neuropeptide K, kassinin, eledoisin, and physalaemin. The detection limit of the assay was 0.07 fmol/100 µL. Samples from the one-probe and the two-probe experiments were analyzed by using two assays. The intra-assay coefficients of variation for 0.1 and 0.5 fmol/100 µL for each assay were 6.2% and 1.2% (one probe) and 4.3% and 2.6% (two probes), respectively. Interassay coefficients of variation for 0.1 and 0.5 fmol/100 µL were 5.6% and 1.8%, respectively.
Histology
We estimated the distribution of the drugs microdialyzed into
the L-7 dorsal horn by dialyzing a 5% ferric chloride solution at the
end of the experiment for 40, 80, or 100 minutes, the times
corresponding to the duration of drug delivery for the dose response
and protocols I and III, respectively. Distilled water was then
dialyzed for 10 minutes to prevent accumulation of the ferric chloride
in the tissue immediately surrounding the probe. The cats were then
euthanatized by administering sodium pentobarbital (120 mg/kg IV). The
section of the spinal cord from L-5 to S-2 was removed and placed in a
solution of 10% phosphate-buffered formalin with 1% potassium
ferrocyanide and potassium ferricyanide and stored at 4°C until
histology was performed. After frozen 50-µm sections of the L-7
dorsal horn were made, anatomic verification of probe placement (22 of
26 cats) and the rostrocaudal distribution of the Prussian blue
reaction (17 of 26 cats) was visualized with a microprojector.
Drugs
All chemicals and drugs were purchased from Sigma Chemical Co.
DAME and naloxone were dissolved in the artificial ECF, pH 7.4. The
lidocaine was dissolved in distilled water. SP was purchased from
Peninsula Laboratories, Inc.
Data Analysis
Data (AP, MAP, HR, and tension) were recorded on an
eight-channel chart recorder. Baseline values were calculated as
averages of the data collected for 1 minute preceding each contraction.
Peak values were calculated for each variable during the 1-minute
contractions and during the first minute of the 5-minute contractions.
A 1-minute interval was used to be consistent with the
contractions of the first part of the study. Developed tension was
calculated as the difference between the resting tension and the peak
tension. For the SP experiments, baseline and peak
cardiovascular and tension values for the two control
contractions from the one-probe experiments were averaged; the same was
done for the two contractions during 200 µmol/L DAME dialysis. Since
data collected from the one-probe and the two-probe experiments were
similar, they were pooled.
All data were initially tested for normality such that appropriate parametric or nonparametric statistical tests could be used. Statistical analysis of the dose-response changes in MAP was performed by using a one-way RM-ANOVA. Baseline and peak values for AP and HR for protocol I, during which 200 µmol/L DAME was microdialyzed in 10 cats, were assessed by using a two-way RM-ANOVA, the factors being time (baseline, peak) and dose (ECF, 40 minutes of 200 µmol/L DAME and 80 minutes of 200 µmol/L DAME). Changes in MAP and HR and developed tension were analyzed by using a one-way RM-ANOVA. The Student-Newman-Keuls test was used for post hoc analysis. For protocol III, comparison of the baseline and peak cardiovascular and SP-LI values, before and after the administration of DAME, was performed by using a two-way RM-ANOVA and the Student-Newman-Keuls test as a post hoc test. Peak changes in MAP, HR, and SP-LI, together with developed tension, before and after the administration of 200 µmol/L DAME, were analyzed by a paired Student's t test or the Wilcoxon signed ranked test when the data were not normally distributed. The significance level was taken as P<.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dose Response
The effects of microdialyzing 20 µmol/L, 200 µmol/L, and 2 mmol/L DAME into the L-7 dorsal horn for
40 minutes are shown in Fig 1
. Although the lowest dose tended to reduce the pressor
response, it was not statistically different from control. The two
highest doses blunted the increase in MAP substantially and had similar
effects. Thus, for all subsequent experiments, we used the 200 µmol/L
dose of DAME dialyzed for 80 minutes, since microdialyzing 200
µmol/L DAME alone for 40 minutes yielded inconsistent
results.
|
Experimental Protocol I
Fig 2 depicts the contraction-induced
cardiovascular responses of a single cat before and
after 80 minutes of 200 µmol/L DAME microdialysis into the L-7
dorsal horn. The pressor and HR responses to the muscle contraction
were attenuated at the 80-minute time period (Fig 2, right). The
mean±SEM baseline and peak values for MAP, HR, and tension during
contraction of the triceps surae as well as the average time for which
the drug was dialyzed are shown in the Table. The mean
tension developed during the contractions and the mean changes in MAP
and HR are depicted in Fig 3. After 49±4 minutes of 200
µmol/L DAME (drug1 in Fig 3, n=9), the changes in MAP and
HR were not different from control. By contrast, both the pressor and
the tachycardiac responses were blunted after 92±3 minutes of
the DAME dialysis (drug2 in Fig 3). The tensions developed
during the control and drug2 contractions were similar. The
developed tension during the drug1 contraction was not
statistically different from control but was greater than that
developed for the drug2 contraction.
|
|
|
Fig 4 portrays a response of a single cat to passive
stretch both before and after microdialysis of 200 µmol/L DAME for
80 minutes into the L-7 dorsal horn. The pressor and HR responses to
the muscle stretch were less after microdialysis of the DAME relative
to the control stretch. The Table
shows the mean baseline and peak
values for MAP, HR, and tension during stretch as well as the average
time for which the drug was dialyzed. Fig 5 depicts the
mean changes in MAP and HR and the tension developed during the
stretches. The pressor and HR responses to stretch after 52±3 minutes
of 200 µmol/L DAME (drug1, n=9) were not different
from control. However, the changes in MAP and HR in response to stretch
after 97±5 minutes of the DAME dialysis (drug2) were less
than both control and drug1 responses. Developed tensions
were equivalent for the three conditions.
|
|
Experimental Protocol II
In three cats, preadministration of naloxone blocked the
effects of DAME on the pressor and HR responses to both muscle
contraction and stretch. The changes in MAP and HR in response to the
control muscle contraction were 40±9 mm Hg and 15±3 beats per
minute, respectively. After microdialyzing 300 µmol/L naloxone for
56±12 minutes into the L-7 dorsal horn, the pressor and HR responses
to muscle contraction were not statistically different from control
(46±16 mm Hg and 17±2 beats per minute, respectively). After
83±1 minutes of 200 µmol/L DAME microdialysis, contraction resulted
in changes in MAP and HR that were similar to both control and
postnaloxone responses (38±11 mm Hg and 14±2 beats per minute,
respectively). Developed tensions were 10.1±0.9, 9.5±0.3, and
8.2±1.4 kg, respectively. One of the three cats developed 50% less
tension during the post-DAME contraction compared with the control
contraction. Nevertheless, the changes in MAP and HR were similar to
those of the control contraction. The control and postnaloxone and
post-DAME pressor and tachycardiac responses to stretch were
comparable (change in MAP, 54±14, 48±13, and 49±12 mm Hg,
respectively; change in HR, 12±4, 13±8, and 9±4 beats per minute,
respectively). Mean developed tensions were identical (9.7±0.6,
9.7±0.6, and 9.7±0.5 kg).
Experimental Protocol III
Fig 6 illustrates the mean changes in MAP and HR as
well as the developed tension for the eight cats used to measure SP
release before and after microdialyzing 200 µmol/L DAME into the L-7
dorsal horn. Also shown in Fig 6 are the mean responses of two cats
that served as time controls. The corresponding changes in SP-LI are
depicted in Fig 7. Despite a slightly larger (not
statistically different) tension being developed after DAME
microdialysis than after no drug dialysis (control), the pressor
response was attenuated. There was no statistical decrease in HR. The
control contraction increased SP-LI from 0.329±0.047 to 0.452±0.071
fmol/100 µL. After 100 minutes of DAME microdialysis, muscle
contraction increased SP-LI from 0.322±0.040 to 0.369±0.042
fmol/100 µL. The change in SP-LI after drug administration was less
than for the control condition (Fig 7).
|
|
In the time-control experiments, similar changes in MAP and HR occurred
for the two contractions (Fig 6). The SP-LI increased from 0.269±0.002
to 0.397±0.012 fmol/100 µL for the first contraction and from
0.216±0.022 to 0.342±0.001 fmol/100 µL for the second
contraction. The changes were similar in magnitude (Fig 7).
Histology
The entire dorsal horn was covered by the Prussian blue reaction,
with the mean rostrocaudal extent for 7 of the 10 cats used in protocol
I being 3.1±0.3 mm and for the 3 cats used in protocol II being
2.9±0.8 mm. Of the 10 cats used in protocol III, 5 had dye dialyzed
into the spinal cord. The area of distribution extended 2.9±0.5 mm
rostrocaudally. In all spinal cords, the probe tract passed through the
center of the L-7 dorsal horn to a depth of 3 mm. The tip of the
probe was at the level of lamina VII. (See Ally et al18
for a representative photomicrograph.)
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using anesthetized cats, we have demonstrated that localized delivery of 200 µmol/L DAME into the L-7 dorsal horn of the spinal cord attenuates the increases in MAP and HR that occur during static muscle contraction and passive stretch. This effect can be blocked by prior administration of naloxone, demonstrating that it is a receptor-specific effect. Further, the inhibitory effect on the contraction-induced pressor response is associated with a reduction in SP release within the dorsal horn. Therefore, these data support the hypothesis that activation of opioid receptors in the L-7 dorsal horn attenuates the cardiovascular responses to static muscle contraction and passive stretch and that the effect may be due to presynaptic inhibition of SP release.
It is well known that group III and IV muscle afferents are activated during muscle contraction and passive stretch.1 2 3 19 20 The group III afferents have been classified as primarily mechanosensitive in nature, although metabosensitive group III afferents also exist; group IV afferents are primarily metabosensitive.19 By inducing a static contraction of the triceps surae muscle, both mechanosensitive and metabosensitive afferents are activated.19 Mainly mechanosensitive afferents are activated during muscle stretch.21 In the present study, both the pressor and HR responses to static contraction and muscle stretch were attenuated by DAME, indicating that opioid receptor activation can influence the activity of both mechanosensitive and metabosensitive afferents.
In the present study, naloxone did not potentiate the pressor response to static contraction or muscle stretch, thus indicating no tonic effect of the opioid inhibitory system. However, in 1987 Go and Yaksh15 reported a naloxone-induced increase in sciatic nerve stimulation–induced SP release from the lumbar spinal cord. This difference could be attributed to different intensities of group III and IV afferent nerve activation, since Jeftinija22 reported in 1988 that naloxone increases afferent-evoked activity at higher but not lower stimulus intensities. Alternatively, naloxone may have potentiated the contraction-induced SP release, but the increased release might not have been sufficient to augment the end-organ response.
That DAME attenuates the pressor and HR response to muscle contraction has been previously reported by Hill and Kaufman10 in 1990. However, there are important differences between the present study and theirs. We report attenuation of the pressor response without any change in baseline MAP. In contrast, Hill and Kaufman only attenuated the pressor response with the intrathecal administration of 100 µg DAME, a dose that reduced baseline MAP by 20%, a statistically significant decrease. This implicates a systemic effect of DAME, since intravenous DAME in cats causes baseline MAP to fall (authors' unpublished data, 1994). Alternatively, the intrathecal DAME may have diffused up to the medulla despite evidence to the contrary. By using microdialysis to deliver the drug into the L-7 dorsal horn, we have reduced the likelihood of systemic or central effects. In addition, we have provided a more localized site of action.
DAME had no effect on the cardiovascular responses to
contraction after 49±4 minutes of microdialysis
(drug1, Fig 3; Table). The tension developed during
the drug1 contraction was greater than that developed after
92±3 minutes of microdialysis (drug2), at which time DAME
blunted the pressor and HR responses to the contraction. We cannot
discount that the higher developed tension may have masked some
attenuation due to the drug. Nevertheless, this does not detract from
the fact that DAME did attenuate the pressor and HR responses after
92±3 minutes of microdialysis. Also, it is our experience that
differences in developed tension of this magnitude within the high
tension range observed in the present study do not lead to vastly
different pressor responses. In addition, it should be noted that no
attenuation was evident during the first post-DAME stretch, the tension
of which was the same as that during the second stretch, at which time
there was significant attenuation.
The presence of opioid receptors in the dorsal horn of the spinal cord has been demonstrated in the rat,23 24 25 the primate,26 and the cat27 28 , with the highest concentration being in laminas I and II.23 27 There appears to be a preponderance of the µ-receptor subtype, the receptor serving DAME.11 Interestingly, opioid receptors are concentrated where most group III and IV afferents originating in the triceps surae muscle terminate, ie, the superficial laminae of the dorsal horn.4 5 In fact, opioid receptors have been identified on primary afferents.11 29 Thus, there appears to be a clear association between opioid receptors and primary afferent neurons in the dorsal roots and the dorsal horns of the spinal cord.
SP is localized in primary afferent neurons and, of particular importance to the present study, has been shown to be present in the substantia gelatinosa of the cat spinal cord.30 31 32 An increase in its release in the lumbar dorsal horn can be induced by noxious stimuli,33 by electrical stimulation of the sciatic nerve at intensities high enough to activate group III and IV primary afferents,15 34 and by static muscle contraction.8 9 18 Since the intrathecal administration of SP-antiserum6 or microinjection of the SP antagonist D-Pro2-D-Phe7-D-Trp9–SP7 both reduce the pressor response to contraction of the triceps surae muscle, there appears to be a role for SP in the transmission of afferent nerve activity from the muscle through the lumbar dorsal horn to the brain stem. The regulation of SP release is the focus of the present study, which demonstrates opioid receptor regulation of contraction-induced SP release.
Static contraction of the triceps surae muscle induced a smaller
increase in SP-LI in the L-7 dorsal horn after 100 minutes of 200
µmol/L DAME relative to the control contraction. The smaller increase
in SP-LI was accompanied by a smaller pressor response. The duration of
the experiments prevented us from testing whether this response was
naloxone reversible. It should be noted, though, that opioid-induced
attenuation of the pressor response to the 1-minute contractions and
stretches of the triceps surae muscle was blocked by naloxone. The
SP-LI and cardiovascular responses to the two
contractions of the time controls were similar to each other, thus
negating the concept of diminishing releasable stores of SP as well as
deterioration of the experimental preparation. Go and
Yaksh15 have shown a naloxone-reversible opioid-induced
(morphine) reduction in the increase in SP-LI from the lumbar spinal
cord in response to sciatic nerve stimulation, and DAME has been shown
to inhibit SP release from primary sensory neurons in
vitro.12 13 35 To the best of our knowledge, this is the
first report of opioid modulation of contraction-induced SP release. We
have recently reported that 2-adrenergic receptor
activation also attenuates the MAP and HR responses to static muscle
contraction and passive stretch, but this attenuation is through a
mechanism other than the presynaptic inhibition of SP
release.18
Although ultrastructural studies have cast doubt as to direct presynaptic opioid interneuron effects,36 37 38 our data support this concept. Indeed, others have also reported opioid-induced presynaptic inhibition of SP release.13 15 35 39 The present study is the first to demonstrate this effect in response to muscle contraction: the DAME probably reduced the release of SP from the group III and IV muscle afferents. The fact that opioid receptors are located on these primary afferents11 29 and that dorsal rhizotomy11 29 40 decreases opioid receptor numbers in the dorsal horn lends further support for a presynaptic inhibitory mechanism involving the muscle afferents. This, however, does not exclude a presynaptic inhibitory effect on the release of SP from SP-containing interneurons or an opioid-induced activation of nonopioidergic interneurons, which then inhibit SP release.
Postsynaptic hyperpolarization of neuronal cell membranes by opioid agonists has been demonstrated.41 42 Therefore, a postsynaptic mechanism may also exist. It is probable, though, that opioids act via both postsynaptic and presynaptic mechanisms.43
The reflex cardiovascular responses to static contraction are thought to be mediated by activation of ergoreceptors.3 44 Ergoreceptors are contraction-sensitive group III and IV afferents that do not behave as nociceptors. Thus, it is possible that the reflex pressor responses and SP-LI release seen in the present study were mediated by muscle afferents that do not encode pain in an awake animal. On the other hand, many group III and IV muscle afferents are nociceptors.3 44 Indeed, SP is thought to be a neurotransmitter/neuromodulator involved in the signaling of pain.45 Opiates are well-known modulators of pain,38 43 and in the present study opiate administration reduced the pressor reflex and attenuated the contraction-induced increase in SP-LI release. Thus, the reflex cardiovascular responses, as well as the increased release of SP-LI, evoked by static contraction may have been influenced by the activation of nociceptors. This point may be particularly salient, since the contractions elicited in the present study were maximal or close to maximal. At least a portion of the muscle ergoreceptors and nociceptors appear to be polymodal, thereby precluding precise categorization for some of the fibers.44 Thus, although the responses demonstrated in the present study are thought to be the result of ergoreceptor activation, we cannot discount the possibility that some of the changes may be mediated by a contraction-induced stimulation of muscle nociceptors.
In conclusion, the present study demonstrates that opioid receptor activation can modulate group III and IV afferent nerve activity passing through the L-7 dorsal horn: the cardiovascular responses to static muscle contraction and stretch of the triceps surae muscle were attenuated. Although our data do not discount a postsynaptic mechanism of action, the attenuation is likely to be a consequence of presynaptic inhibition of SP release, probably from the activated muscle afferents.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by National Heart, Lung, and Blood Institute program project HL-06296 and the Lawson and Rogers Lacy Research Fund in cardiovascular diseases. Dr Meintjes is a recipient of an individual national research service award (1-F32-HL-08967-01). Dr Nóbrega was supported by Capes Brazil (No. 2132/92-3). We appreciate the expert technical assistance of James Jones and Julius Lamar.
|
Footnotes |
|---|
Previously published as preliminary data in abstract form (Neuroscience. 1993;19:1483).
Received January 17, 1995; accepted April 26, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Coote JH, Hilton SM, Perez-Gonzalez JF. The reflex nature of the pressor response to muscular exercise. J Physiol (Lond). 1971;215:789-804.
2.
McCloskey DI, Mitchell JH. Reflex
cardiovascular and respiratory responses originating in
exercising muscle. J Physiol (Lond). 1972;224:173-186.
3. Mitchell JH, Schmidt RF. Cardiovascular reflex control by afferent fibers from skeletal muscle receptors. In: Shepherd JT, Abboud FM, eds. Handbook of Physiology, Section 2: The Cardiovascular System, Volume III. Bethesda, Md: American Physiological Society; 1983:623-658.
4. Kalia M, Mei SS, Kao FF. Central projections from ergoreceptors (C fibers) in muscle involved in cardiopulmonary responses to static exercise. Circ Res. 1981;48(suppl I):I-48-I-62.
5. Mense S, Craig AD. Spinal and supraspinal terminations of primary afferent fibers from the gastrocnemius-soleus muscle in the cat. Neuroscience. 1988;26:1023-1035. [Medline] [Order article via Infotrieve]
6. Kaufman MP, Rybicki KJ, Kozlowski GP, Iwamoto GA. Immunoneutralization of substance P attenuates the reflex pressor response to muscular contraction. Brain Res. 1986;377:199-203. [Medline] [Order article via Infotrieve]
7.
Wilson LB, Wall PT, Matsukawa K, Mitchell JH.
Effect of spinal microinjections of an antagonist to
substance P or somatostatin on the exercise pressor reflex.
Circ Res. 1992;70:213-222.
8.
Wilson LB, Fuchs IE, Matsukawa K, Mitchell JH, Wall
PT. Substance P release in the spinal cord during the exercise
pressor reflex in anaesthetized cats. J Physiol
(Lond). 1993;460:79-90.
9.
Wilson LB, Fuchs IE, Mitchell JH. Effects of
graded muscle contractions on spinal cord substance P release,
arterial blood pressure, and heart rate.
Circ Res. 1993;73:1024-1031.
10.
Hill JM, Kaufman MP. Attenuation of reflex
pressor and ventilatory responses to static muscular contraction by
intrathecal opioids. J Appl
Physiol. 1990;68:2466-2472.
11. Fields HL, Emson PC, Leigh BK, Gilbert RFT, Iversen LL. Multiple opiate receptor sites on primary afferent fibers. Nature. 1980;284:351-353. [Medline] [Order article via Infotrieve]
12.
Mudge AW, Leeman SE, Fischbach GD. Enkephalin
inhibits release of substance P from sensory neurons in culture and
decreases action potential duration. Proc Natl Acad Sci
U S A. 1979;76:526-530.
13.
Chang HM, Berde CB, Holz GG, Steward GF, Kream RM.
Sufentanil, morphine, met-enkephalin, and 
-agonist
(U-50,488H) inhibit substance P release from primary sensory neurons: a
model for presynaptic spinal opioid actions.
Anesthesiology. 1989;70:672-677. [Medline]
[Order article via Infotrieve]
14. Yaksh TL, Jessell TM, Gamse R, Mudge AW, Leeman SE. Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo. Nature. 1980;286:155-157. [Medline] [Order article via Infotrieve]
15.
Go VL, Yaksh TL. Release of substance P from the
cat spinal cord. J Physiol (Lond). 1987;391:141-167.
16. Wilson LB, Wall PT, Matsukawa K, Mitchell JH. Multiplicity of the afferent pathways mediating the exercise pressor reflex. Brain Res. 1991;539:316-319.[Medline] [Order article via Infotrieve]
17.
Pert CB, Pert A, Chang J-K, Fong BT.
[D-Ala2]-Met-enkephalinamide: a potent,
long-lasting synthetic pentapeptide analgesic.
Science. 1976;194:330-332.
18.
Ally A, Meintjes AF, Mitchell JH, Wilson LB.
Effects of clonidine on the reflex
cardiovascular responses and release of substance P
during muscle contraction. Circ Res. 1994;75:567-575.
19.
Kaufman MP, Longhurst JC, Rybicki KJ, Wallach JH,
Mitchell JH. Effects of muscular contraction on impulse activity
of groups III and IV afferents in cats. J Appl
Physiol. 1983;55:105-112.
20. Kaufman MP, Waldrop TG, Rybicki KJ, Ordway GA, Mitchell JH. Effects of static and rhythmic twitch contractions on the discharge of group III and IV muscle afferents. Cardiovasc Res. 1984;28:663-668.
21.
Stebbins CL, Brown B, Levin D, Longhurst JC.
Reflex effect of skeletal muscle mechanoreceptor stimulation on
the cardiovascular system. J
Appl Physiol. 1988;65:1539-1547.
22. Jeftinija S. Enkephalins modulate excitatory synaptic transmission in the superficial dorsal horn by acting at µ-opioid receptor sites. Brain Res. 1988;460:260-268. [Medline] [Order article via Infotrieve]
23. Atweh SF, Kuhar MJ. Autoradiographic localization of opiate receptors in rat brain, I: spinal cord and lower medulla. Brain Res. 1977;124:53-67. [Medline] [Order article via Infotrieve]
24.
Goodman RR, Snyder SH, Kuhar MJ, Young WS.
Differentiation of delta and mu opiate receptor localizations by
light microscopic autoradiography. Proc
Natl Acad Sci U S A. 1980;77:6239-6243.
25.
Slater P, Patel S. Autoradiographic
localization of opiate receptors in the rat spinal cord.
Eur J Pharmacol. 1983;92:159-160. [Medline]
[Order article via Infotrieve]
26. Lamotte C, Pert CB, Snyder SH. Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section. Brain Res. 1976;112:407-412. [Medline] [Order article via Infotrieve]
27. Dashwood MR, Gilbey MP, Spyer KM. The localization of adrenoceptors and opiate receptors in regions of the cat central nervous system involved in cardiovascular control. Neuroscience. 1985;15:537-551. [Medline] [Order article via Infotrieve]
28. Pollard H, Bouthenet ML, Moreau J, Souil E, Verroust P, Ronco P, Schwartz JC. Detailed immunoautoradiographic mapping of enkephalinase (EC 3.4.24.11) in rat central nervous system: comparison with enkephalins and substance P. Neuroscience. 1989;30:339-376. [Medline] [Order article via Infotrieve]
29. Ninkovic M, Hunt SP, Kelly JS. Effect of dorsal rhizotomy on the autoradiographic distribution of opiate and neurotensin receptors and neurotensin-like immunoreactivity within the rat spinal cord. Brain Res. 1981;230:111-119. [Medline] [Order article via Infotrieve]
30.
Hökfelt T, Kellerth JO, Nilsson G, Pernow B.
Substance P: localization in the central nervous system and in
some primary sensory neurons. Science. 1975;190:889-890.
31. Barber RP, Vaughn JE, Slemmon JR, Salvaterra PM, Roberts E, Leeman SE. The origin, distribution and synaptic relationships of substance P in rat spinal cord. J Comp Neurosci. 1979;184:331-352.
32. McNeill DL, Westlund KN, Coggeshall RE. Peptide immunoreactivity of unmyelinated primary afferent axons in rat lumbar dorsal roots. J Histochem Cytochem. 1989;37:1047-1052. [Abstract]
33. Kuraishi Y, Hirota N, Sato Y, Hanashima N, Takagi H, Satoh M. Stimulus specificity of peripherally evoked substance P release from the rabbit dorsal horn in situ. Neuroscience. 1989;30:241-250. [Medline] [Order article via Infotrieve]
34. Brodin E, Linderoth B, Gazelius B, Ungerstedt U. In vivo release of substance P in cat dorsal horn studied with microdialysis. Neurosci Lett. 1987;76:357-362. [Medline] [Order article via Infotrieve]
35. Jessell TM, Iversen LL. Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature. 1977;268:546-551.
36. Hunt SP, Kelly JS, Emson PC. The electron microscopic localization of methionine-enkephalin within the superficial layers (I and II) of the spinal cord. Neuroscience. 1980;5:1871-1890. [Medline] [Order article via Infotrieve]
37. Sumal KK, Pickel VM, Miller RJ, Reis DJ. Enkephalin-containing neurons in the substantia gelatinosa of spinal trigeminal complex: ultrastructure and synaptic interaction with primary sensory afferents. Brain Res. 1982;248:223-236. [Medline] [Order article via Infotrieve]
38. Glazer EJ, Basbaum AI. Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn. Neuroscience. 1983;10:357-376. [Medline] [Order article via Infotrieve]
39.
MacDonald RL, Nelson PG. Specific-opiate-induced
depression of transmitter release from dorsal root ganglion cells in
culture. Science. 1978;199:1449-1450.
40.
Zajac J-M, Lombard M-C, Peschanski M, Besson J-M,
Roques BP. Autoradiographic study of µ and 
opioid binding sites and neutral endopeptidase-24.11 in
rat after dorsal root rhizotomy. Brain Res. 1989;477:400-403. [Medline]
[Order article via Infotrieve]
41. Zeiglgänsberger W, Bayerl H. The mechanism of inhibition of neuronal activity by opiates in the spinal cord of cat. Brain Res. 1976;115:111-128. [Medline] [Order article via Infotrieve]
42. Yoshimura M, North RA. Substantia gelatinosa neurones hyperpolarized in vitro by enkephalin. Nature. 1983;305:529-530. [Medline] [Order article via Infotrieve]
43. Lombard M-C, Besson J-M. Attempts to gauge the relative importance of pre- and postsynaptic effects of morphine on the transmission of noxious messages in the dorsal horn of the rat spinal cord. Pain. 1989;37:335-345. [Medline] [Order article via Infotrieve]
44. Kniffeki KD, Mense S, Schmidt RF. Muscle receptors with fine afferent fibers which may evoke circulatory reflexes. Circ Res. 1981;48(suppl I):I-25-I-31.
45. Pernow B. Substance P. Pharmacol Rev. 1983;35:85-141.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  P. M. Schmitt and M. P. Kaufman Estrogen's attenuating effect on the exercise pressor reflex is more opioid dependent in gonadally intact than in ovariectomized female cats J Appl Physiol, February 1, 2005; 98(2): 633 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Wilson, J. Engbretson, and A. D. Crews Pressor reflex evoked by static muscle contraction: role of nitric oxide in the dorsal horn Am J Physiol Regulatory Integrative Comp Physiol, June 1, 1999; 276(6): R1639 - R1646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Wilson and G. A. Hand Segmental effect of spinal NK-1 receptor blockade on the pressor reflex Am J Physiol Heart Circ Physiol, September 1, 1998; 275(3): H789 - H796. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||