1024
Effects of Graded Muscle Contractions on
Spinal Cord Substance P Release, Arterial
Blood Pressure, and Heart Rate
L. Britt Wilson, Ingbert E. Fuchs, Jere H. Mitchell
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The release of substance P (SP)-like immunoreactivity (SP-LI) in the dorsal horn of the spinal cord and
the cardiovascular changes to both high-tension (HT) and low-tension (LT) contractions were determined
using c-chloralose-anesthetized cats. Over a 10-minute period, seven contractions (HT or LT) were
induced. Each contraction was 20 seconds in duration and was followed by an 80-second quiescent period.
The tension-time index (TTI) for the HT contractions was 2751±348 kg * s (mean± SD), which was greater
than the TTI of 813±167 kg s for the LT contractions. The HT contractions caused a greater release of
SP-LI than the LT contractions: SP-LI increased from 0.18±0.02 to 0.32±0.03 fmol/100 ,uL and from
0.18±0.02 to 0.25±0.04 fmol/100 ,L for the two types of contractions, respectively. Concomitant with this
greater SP-LI release, HT contractions caused larger increases in mean arterial pressure (34±16 versus
11±4 mm Hg) and heart rate (18±7 versus 8±4 beats per minute) than did the LT contractions. These
changes in SP-LI, mean arterial pressure, and heart rate were virtually abolished when the contractions
were repeated after sectioning the L-5-S-2 dorsal and ventral roots or when the electrical stimulation of
the ventral roots was repeated after muscle paralysis with gallamine triethiodide. These results
demonstrate that contraction-evoked SP-LI release in the dorsal horn is related to the developed tension.
Furthermore, these data provide additional support for the hypothesis that the release of SP from the
central terminations of muscle afferents plays a role in mediating the cardiovascular responses to static
contraction of skeletal muscle. (Circ Res. 1993;73:1024-1031.)
KEY WORDs * substance P * exercise pressor reflex * cats * dorsal horn * microdialysis
It has been demonstrated using anesthetized cats
that static contraction of the triceps surae muscle
increases arterial blood pressure, heart rate, myocardial contractility, and renal sympathetic nerve activity.1-6 These changes are a reflex arising from the
contracting muscle, because they are abolished by cutting the appropriate spinal roots.23,5,6 The reflex cardiovascular and sympathetic responses are mediated by a
contraction-induced activation of group ILL (thinly myelinated) and group IV (unmyelinated) muscle afferents.2 These afferents appear to respond to the mechanical and metabolic alterations that occur during the
contraction.7-9 Furthermore, the magnitude of afferent
activation and the reflex autonomic responses are related to the developed tension.1,5,8,40411
The majority of group III and IV fibers synapse in the
dorsal horn of the spinal cord.12-14Thus, this region may
be the site of the first synapse for the reflex cardiovascular changes seen during muscle contraction. The
dorsal horn of the spinal cord contains numerous compounds that may be the neurotransmitters/neuromoduReceived May 21, 1993; accepted August 9, 1993.
From the Moss Heart Center and Department of Physiology,
University of Texas Southwestern Medical Center, Dallas.
Previously presented as a preliminary report in abstract form
(FASEB J. 1992;6:A1168).
Correspondence to L. Britt Wilson, PhD, Moss Heart Center,
University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd, Dallas, TX 75235-9034.
lators of primary afferents.15-17 Substance P (SP) is one
such compound.18-20
Several studies have investigated the possibility that
the release of SP in the dorsal horn plays a role in
mediating the reflex changes elicited by static contraction.21-25 Administration of an antagonist to SP, either
intrathecally or directly into the dorsal horn, blunts the
cardiovascular changes elicited by static contraction.21,24
Hill et a123 showed that the intrathecal administration of
a neurokinin-1 (NK-1; this receptor preferentially binds
SP) antagonist attenuates the pressor response elicited
by muscle contraction. Furthermore, we have recently
demonstrated that static muscle contraction increases
the release of SP in the dorsal horn.25 Thus, the release
of SP in the spinal cord as a result of muscle afferent
activation appears to play a role in mediating the reflex
cardiovascular and renal sympathetic increases caused
by static contraction. Because the reflex autonomic
changes are related to the developed tension,1,5,1011 we
hypothesized that the release of SP in the dorsal horn is
also influenced by the degree of force production.
Materials and Methods
Experimental Preparation
Mongrel male cats (mean weight, 3.9 kg; n=8) were
anesthetized by breathing a gaseous mixture containing
halothane (2% to 5%), nitrous oxide (1 to 3 L/min), and
oxygen (1 to 3 L/min). An endotracheal tube was
inserted into the airway via a tracheotomy, and cathe-
Wilson et al Muscle Tension and Substance P Release
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ters were placed into a jugular vein and a carotid artery.
After catheterization, the anesthetic gas mixture was
removed, and anesthesia was maintained with a-chloralose (60 mg/kg IV). If the animal's pupils became
dilated and/or they exhibited a corneal reflex or withdrawal of the limb in response to noxious pinch of the
paw, additional a-chloralose (5 to 10 mg/kg IV) or
sodium pentobarbital (1 to 2 mg/kg) was administered
to maintain an adequate depth of anesthesia. In those
animals that were paralyzed (see below), additional
anesthesia was provided if arterial blood pressure
and/or heart rate increased after inducing the paralysis.
The lungs were ventilated with room air using a respirator (model 552, Harvard Instruments, South Natick,
Mass). Arterial blood gases and pH were periodically
checked and maintained within normal limits (pH, 7.30
to 7.40; Pco2, 32 to 36 mm Hg; Po2, >80 mm Hg) by
adjusting the ventilator, providing supplemental oxygen,
or injecting sodium bicarbonate intravenously. Rectal
temperature was continuously monitored and maintained between 370 and 38.5°C by a water-perfused
heating pad and an external heat lamp.
A laminectomy was performed exposing the lower
lumbar and upper sacral portions of the spinal cord.
The dura was opened, and the spinal roots were visually
identified. The L-7 and S-1 dorsal and ventral roots
were carefully separated, and the ventral roots were cut
close to the spinal cord. The L-5, L-6, and S-2 dorsal
and ventral roots were sectioned. Cutting these roots
should have no effect on the reflex cardiovascular
responses induced by static contraction.26 The calcaneal
bone was cut, allowing the Achilles tendon to be connected to a force transducer (model F10, Grass Instrument Co, Quincy, Mass). The pelvis was stabilized in a
spinal unit (David Kopf Instruments, Tujunga, Calif),
and the knee joint was secured by attaching the patellar
tendon to a steel post. The exposed spinal cord region
was kept moist with saline.
Arterial blood pressure was measured by connecting
the carotid artery catheter to a pressure transducer
(model P231D, Statham, Oxnard, Calif). Mean arterial
pressure was obtained by integrating the arterial signal
with a time constant of 4 seconds. Heart rate was
derived from the arterial pressure pulse by a biotachometer (Gould Instruments, Cleveland, Ohio), and muscle
tension was measured using the force transducer. The
tension-time index (TTI) for each contraction was
determined by measuring the area under the tension
trace. The TTI for the high-tension (HT) or low-tension
(LT) contractions represents the sum of the TTI values
for the 14 contractions (see below). All measured
variables were continuously recorded on an eight-channel chart recorder (Gould, 2800S). Control values were
determined by analyzing at least 30 seconds of the data
immediately before muscle contraction.
Microdialysis
After isolating the dorsal and ventral roots, a microdialysis probe (model CMA-5410, 3 mm membrane, Bioanalytical Systems Inc, West Lafayette, Ind) was lowered into the center of the rostral-caudal extent of the
L-7 dorsal horn region using a stereotaxic carrier (David Kopf Instruments). The probe was inserted so that
the entire membrane was submerged in the spinal cord
tissue. The probe was continuously perfused at a rate of
1025
5 ,uL/min with a physiological salt solution 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). This
solution was made fresh for each experiment (in vivo
and in vitro, see below). After probe insertion, timed
collections (20 minutes) were begun. During the six
collection periods, the probe was surrounded by agar so
that the height of the agar mound was even with the
muscles of the back. Next, a piece of parafilm was
placed around the shaft of the probe, on top of the agar.
A quick-setting silicone elastomer (A643-K, Smith and
Nephew Rolyan Co, Menomonee Falls, Wis) was applied to the top of the parafilm so that the parafilm was
fixed to the shaft of the probe and the muscles of the
back. This procedure allowed the carrier to be removed
from the shaft of the probe; thus, the probe remained in
the same relative position despite any movement of the
back. On a separate day, the concentration of SP-like
immunoreactivity (SP-LI) of the perfusates was measured using a radioimmunoassay (RIA; see below). The
RIA was performed in a blinded fashion.
A new probe was used for each experimental animal.
The percent recovery of SP-LI for five of the eight
probes was tested in vitro on a separate day. The probe
was perfused at the same rate as used for the animal
experiment (5 ,uL/min). The probe was inserted into
vials containing varying concentrations of SP. A minimum of three timed collections (20 minutes) was taken
from each vial, and the percent recovery from the first
collection was omitted to ensure that stabilization of the
concentration gradient occurred. The average percent
recovery from the remaining two collections was calculated to determine the percent recovery for each probe.
For three of the probes, this in vitro test was performed
before use in the animal. The remaining two probes
were tested after the animal experiment. The recovery
for the three probes that were tested before the experiment was 12.3+1.9% (mean±SD), which was similar to
the other two probes (14.4±1.7%). The average recovery of SP-LI for the five probes was 13.1+2.0%.
RIA for SP-LI
This procedure has been previously described in
detail.25 Briefly, removable microplate wells (Removawell Immulon 4, Dynatech, Rochester, NY) were
coated with 1 ,g purified protein A (Purified Recomb
Protein A, binding 1.3 mg rabbit IgG per mg, Pierce
Chemical Co, Rockford, Ill) in 100 ,uL 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 again
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 phosphate-buffered
saline overnight at 4°C. After washing three times with
the RIA buffer, standards in triplicate and samples (90
,uL) were added to the wells and incubated for 24 hours
at 40C. '251-labeled [Tyr8]SP-(1-11) (110 ,uL) was added
to standards and samples and incubated for 24 hours at
40C. Wells were washed three times with RIA buffer,
separated, and counted for 10 minutes in a 20-well
1026
Circulation Research Vol 73, No 6 December 1993
TABLE 1. Microdialysis Protocol
Collection Period
After insertion of probe
Contractions (HT or LT, 7 total)
After contraction
Repeat contractions
After contraction
Contractions (HT or LT, 7 total)
After contraction
Repeat contractions
After contraction
Collection Time, min
6x20
10
2x20
10
2x20
10
2 x20
10
2 x20
HT indicates high tension; LT, low tension.
gamma
counter, using
a
cubic spline algorithm for data
processing.
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The cross-reactivity at IC50 was 100% for SP-(1-11)
and SP-(2-11); 98% for 125[Tyr8]SP; 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); <0.01% for SP-(7-11), SP-(8-11), SP-(911), neurokinin A (NKA), neurokinin B (NKB), somatostatin, neuropeptide K, kassinin, eledoisin, and
physalaemin. The detection limit of the assay was 0.07
fmol/100 ,L, and the mean±SD for the IC20, IC50, and
IC80 was 0.32±0.08, 0.98±0.12, and 3.80± 1.3 fmol/100
,uL, respectively.
Experimental Protocol
After inserting the microdialysis probe, six 20-minute
collection periods (flow rate, 5 ,uL/min) were performed
(Table 1). These control collections were performed so
that basal SP-LI release was attained before any muscle
manipulation.2728 After a sample was collected, it was
stored in a freezer (-80°C) until the assay was performed. The probe was continuously perfused throughout the experiment.
Once the control collections were completed, a series
of HT or LT contractions was evoked during a 10minute collection period (Table 1). A total of seven
contractions were evoked for each of the 10-minute
collection periods. The duration of each contraction was
20 seconds, and each was separated by an 80-second
quiescent period. The HT contractions were elicited by
simultaneously stimulating the peripheral ends of the
cut L-7 and S-1 ventral roots (3x motor threshold, 40
Hz, 0.1 millisecond) with a resting tension of 1.0 kg set
on the muscle. LT contractions were evoked by stimulating only the L-7 ventral root (1.5 to 2.Ox motor
threshold, 40 Hz, 0.1 millisecond), and resting tension
was set at 200 g. Before the electrical stimulation, the
ventral roots were placed on the electrodes, and the
saline covering the spinal cord was removed. Thus, only
air surrounded the ventral roots while they were stimulated. The contractions began approximately 20 seconds after the onset of the 10-minute collection period.
Next, the muscle was allowed to recover during two
20-minute collection periods. This pattern was repeated
until four sets of contractions were performed. Thus, a
total of 28 contractions were performed (14 HT and 14
LT contractions). The dialysates from the HT contrac-
tions were then combined so that the sample volumes
remained equal. Likewise, the dialysates from the LT
contractions were combined. Whether the HT or LT
contractions were performed first was randomized.
However, the contractions always occurred consecutively; ie, if the HT contractions were performed first,
then the next series of contractions was HT. This
protocol was performed on all the cats.
After the aforementioned procedure had been accomplished, one of two different protocols was performed on a given cat. The L-7 and S-1 dorsal roots
were cut on some of the cats (n=5), and then the
protocol described in Table 1 was repeated. This was
done to test if the neuropeptide release was the result of
activation of muscle afferents. For three of the animals,
the L-7 and S-1 ventral roots were stimulated while the
cat was paralyzed (3 mg/kg gallamine triethiodide) to
determine if changes in peptide release were the result
of muscle contraction and not current spread associated
with the electrical stimulus. The electrical parameters
for this test were 5 x motor threshold, 50 Hz, and 0.1
millisecond, which were greater than those used for the
HT contractions (see above). The animals were euthanatized (120 mg/kg sodium pentobarbital IV), and the
L-6-S-1 region of the spinal cord was removed and
placed in 10% formalin for tissue fixation. Once the
tissue was adequately fixed, 50-,gm frozen (transverse)
sections of the L-7 region of the spinal cord were cut
using a cryostat (2800 Frigocut E, Reichert-Young,
Cambridge Instruments, Buffalo, NY) to determine the
location of the tract caused by the microdialysis probe.
Statistical Analysis
All data are expressed as mean±SD. SP-LI values
during the six precontraction collections were analyzed
using a one-way ANOVA.29 The ANOVA was also used
to compare the absolute concentration of the precontraction and HT and LT contraction SP-LI values (Fig
2). Tukey's test was performed when a significant F
value was found. For the SP-LI, hemodynamic, and
tension data, a paired Student's t test was performed to
compare the changes elicited by the HT versus LT
contractions. For all analyses, P<.05 was used as the
level of statistical significance.
Results
The concentration of SP-LI for the six precontraction
collections is illustrated in Fig 1. Initially, SP-LI was
relatively high but decreased thereafter. The SP-LI
values during the last two collections were essentially
identical, suggesting that this was the basal release for
this preparation. The last bar represents the precontraction SP-LI concentration for the HT or LT contractions
that were subsequently performed. Both HT and LT
contractions increased SP-LI release in the dorsal horn
of the spinal cord (Fig 2). However, the HT contractions
evoked a larger increase in SP-LI compared with the LT
contractions.
An original recording from one cat illustrating the
cardiovascular responses to the HT and LT contractions
is shown in Fig 3. During a 10-minute collection period,
a series of seven HT contractions was evoked (upper
panel of Fig 3). During a subsequent 10-minute collection period, a series of seven LT contractions was
elicited (lower panel of Fig 3). This recording demon-
Wilson et al Muscle Tension and Substance P Release
0.40-
0.30
l.
8
.-
qT
t 0.20
u0
0.10*
U.UU1
L
T
-
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8
0
3
m
3
3
-,
3u
3
m
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Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
FIG 1. Bar graph showing the concentration of substance P-like immunoreactivity (SP-LI) during the six
timed (20-minute) collections after inserting the microdialysis probe into the L-7 dorsal horn of the spinal cord.
Data are mean+SD (n=8). PI indicates postinsertion (of
the microdialysis probe). *Significant difference compared with the first bar (0-20 min PI).
strates that the HT contractions caused larger increases
in mean arterial pressure and heart rate compared with
the LT contractions. The average data for TTI, change
in SP-LI, and hemodynamic responses to the HT and
LT contractions are illustrated in Fig 4. As designed, the
HT contractions produced a higher TTI compared with
the LT contractions (Fig 4A). A greater SP-LI release
occurred in response to this larger TTI (Fig 4B).
Likewise, there were greater increases in mean arterial
pressure (Fig 4C) and heart rate (Fig 4D) in response to
the HT contractions. Thus, the release of SP and the
cardiovascular responses to static contraction are related to the developed tension. The precontraction
hemodynamic variables were not different (Table 2).
The relation between TTI, change in SP-LI, and
change in mean arterial pressure is depicted in Fig 5.
The upper and middle graphs show that the change in
SP-LI and the change in mean arterial pressure are
a
Preo-ontracton
HT Contractons
LT Contractions
T
8
(0
U."
FIG 2. Graph illustrating the concentration of substance
P-like immunoreactivity (SPLI) before (open bars) and
during high-tension (HT) contractions (filled bar) and
low-tension (LT) contractions (hatched bar). Data are
mean+SD (n=8). *Significant difference compared with
precontraction values. 'Significant difference compared
with HT contractions.
1027
related to the TTI. A relation exists between the change
in SP-LI and the change in mean arterial pressure, but
it appears to be weaker than the previous relations, as
indicated by the overlap of the deviation bars (Fig 5,
lower graph).
The precontraction and contraction SP-LI values
before and after cutting the L-7 and S-1 dorsal roots for
the five cats in which this procedure was performed are
depicted in the upper panel of Fig 6. Before sectioning
the dorsal roots, both HT and LT contractions increased SP-LI release. However, these increases were
virtually abolished after cutting the L-7 and S-1 dorsal
roots. Thus, the changes in SP-LI release in the dorsal
horn in response to static contraction are the result of
muscle afferent activation. The lower panel of Fig 6
illustrates that the contraction-induced release of SP-LI
is abolished if the contraction is prevented by muscle
paralysis. This indicates that the changes in SP-LI
release demonstrated in this study are not the result of
direct activation of muscle afferents by the electrical
stimulus.
The position of the probe was determined histologically. The results verified that the probe was in the
dorsal horn. The central canal was the approximate
lateral level of the tip of the membrane, ie, in the
ventral portion of lamina VII or the most dorsal aspect
of lamina VIII. This is similar to the location found in
our previous study and suggests that the extracellular
space of the entire dorsal horn was probably sampled.25
Discussion
The purpose of this study was to determine if the
contraction-induced increases in the release of SP in the
dorsal horn, as well as the cardiovascular changes, are
related to the developed tension. The developed tension
was much higher for the HT compared with the LT
contractions. The reflex cardiovascular changes were
related to force production, a finding that has been
shown previously.1,5,10,11 An additional observation was
that HT contractions elicited greater increases in the
release of SP in the dorsal horn of the spinal cord
compared with the LT contractions. Thus, the release of
SP in the dorsal horn from muscle afferents is related to
the developed tension.
Using microdialysis, Brodin et a130 demonstrated that
electrical stimulation of the sciatic nerve increases SP
release in the dorsal horn of anesthetized cats. However, this increase only occurred if the intensity of the
stimulus was such that it activated group III and IV
afferents. A similar finding was reported by Go and
Yaksh,31 who used an in vitro spinal cord preparation.
Thus, SP may play a role in sensory transmission at the
level of the spinal cord. Along these lines, several
studies have provided evidence that the spinal release of
SP plays a role in mediating the reflex cardiovascular
responses to muscle contraction.21-25 The present study
provides additional support that the release of SP from
muscle afferents is involved in the reflex autonomic
changes produced by static contraction.
SP is a member of a class of neuropeptides collectively referred to as tachykinins.3233 The other members
of this class found in mammals are NKA and NKB.
Three different receptors mediate the actions of these
tachykinins: NK-1, NK-2, and NK-3.33 The actions of SP
are thought to be mediated by the NK-1 receptor,
Circulation Research Vol 73, No 6 December 1993
1028
HIGH TENSION
HR
(bwWn)
AP
AP
lln
20
LOW TENSION
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0
-
To~
LW
20
SeW
FIG 3. Original tracings from one cat demonstrating the tension and cardiovascular responses to a series of high-tension
contractions (top) and low-tension contractions (bottom). HR indicates heart rate; AP, arterial pressure.
whereas NKA preferentially binds to NK-2. SP and
NKA are found in the afferents synapsing in the dorsal
horn, and in some of these fibers, the neuropeptides
may be colocalized.1819,34,35 Because of this, it may be
speculated that NKA is involved in the reflex responses
to muscle contraction. Duggan et a136 demonstrated that
HT Contractions
30001
r
I
^ o
LT Contracions
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0
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muscle contraction increases NKA release in the dorsal
horn of the spinal cord. However, Hill et a123 showed
that injecting an NK-2 antagonist into the intrathecal
space of the lumbar spinal cord fails to alter the reflex
pressor response to muscle contraction. Injection of an
NK-1 antagonist blunted these reflex changes. Thus, the
possible role of NKA in the reflex responses to muscle
contraction is unclear. Any confounding influence of
NKA release in this study is unlikely, because the
antibody used in the RIA showed virtually no crossreactivity to NKA.
There appeared to be a relation between TTI and the
change in mean arterial pressure (Fig 5). Previous
studies have shown that the reflex cardiovascular responses to static muscle contraction are related to the
developed tension.,10°1' There is also a tendency for the
reflex changes in renal sympathetic nerve activity
evoked by static contraction to be related to the developed tension.5 Using anesthetized cats, Perez-Gonzalez1l examined the relation of the arterial blood pressure changes to three different indexes of tension: (1)
peak tension, (2) mean tension, and (3) TTI. The
TABLE 2. Precontraction Hemodynamic Data
FIG 4. Bar graphs depicting the tension-time index (TTI,
A), change in substance P-like immunoreactivity (SP-LI,
B), change in mean arterial pressure (MAP, C), and the
change in heart rate (HR, D) in response to high-tension
(HT) contractions (open bars) and low-tension (LT) contractions (filled bars). Data are mean-+-SD (n=8). *Significant difference compared with HT contractions.
HT Contractions
LT Contractions
143±14
MAP, mm Hg
137+10
204+36
HR, bpm
209+26
HT indicates high tension; LT, low tension; MAP, mean arterial
pressure; HR, heart rate; and bpm, beats per minute. Values are
mean+SD (n=8).
1
Wilson et al Muscle Tension and Substance P Release
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Post-Paralysis
Chango in SP-LI (fmol1OO 1l)
FIG 5. Top and middle, Graphs illustrate the relation of
tension-time index (TTI) and the change in substance
P-like immunoreactivity (SP-LI) and TTI and the change
in mean arterial pressure (MAP) for low-tension (LT)
contractions (A) and high-tension (HT) contractions (A).
Bottom, Graph depicts the relation of the change in SP-LI
and the change in MAP. Symbols are the same as for the
upper two graphs. Data are mean+SD (n=8).
FIG 6. Top, Bar graph shows the substance P-like
immunoreactivity (SPLI) values before (open bars) and
during high-tension (HT) contractions (filled bar) and
low-tension (LT) contractions (hatched bar) before and
after cutting the L-7 and S-1 dorsal root (DR) for the five
cats in which this was performed. Bottom, The first four
bars of the bar graph illustrate the concentration of SPLI
before (open bars) and during HT contractions (filled bar)
and LT contractions (hatched bar) before muscle paralysis for the three cats in which this was performed. The
second two bars represent the SPLI values before (open
bar) and during (cross-hatched bar) electrical stimulation
of the L-7 and S-1 ventral roots (VR). Data are mean ±SD.
strongest relation that existed (r=0.87) was for TTI.
Further, this correlation between the pressor responses
and TTI only existed for tetanic, not rhythmic, contractions. In the present study, only tetanic contractions
were evoked. Thus, the relation between the change in
mean arterial pressure and TTI found in this study is in
agreement with Perez-Gonzalez.
A new finding from the present study is that the
release of SP in the dorsal horn of the spinal cord is also
related to the TTI (Fig 5). The discharge of mechanically sensitive muscle afferents is related to developed
tension.8 Although not tested, it is quite plausible that
the discharge of metabolically sensitive afferents is
related to TTI, since muscle oxygen consumption is
predominately influenced by this parameter.37 Further,
it appears that activation of metabolically sensitive
muscle afferents is necessary to evoke SP release in this
model, since microinjecting an antagonist to this neuropeptide has no effect on the reflex responses elicited
by muscle stretch.24 Thus, the relation between SP
release and TTI provides further support for the hypothesis that the release of this neuropeptide into the
dorsal horn plays a role in mediating the reflex cardiovascular responses to static muscle contraction.
On the other hand, because of the rather large
deviation bars, the relation between the change in SP-LI
and the change in mean arterial pressure appears weak
(Fig 5, lower graph). This is not surprising, since the
release of SP from muscle afferents represents the first
synapse in the reflex arc. Because an intact medulla is
required for full expression of the cardiovascular responses to muscle contraction,38 a minimum of two
more synapses in the central nervous system must be
involved in this reflex. Thus, multiple sites exist for
integration and/or modulation of the signal arising from
the second-order neuron. Further, the exact relation
between neurally released SP and the discharge of
dorsal horn cells is unknown. This is also complicated by
the fact that SP does not evoke fast excitatory postsynaptic potentials when applied to dorsal horn cells.39.40
Thus, it may be quite difficult to demonstrate a strong
relation between spinal cord SP release and the end
organ response (change in mean arterial pressure).
However, this should not distract from the relation
between SP release and the change in mean arterial
pressure for the two levels of tension that is illustrated
in Fig 5. In our previous study,25 cutting the L-6-S-1
dorsal and ventral roots markedly attenuated, but did
not abolish, contraction-induced SP release. In the
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Circulation Research Vol 73, No 6 December 1993
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present study, cutting the L-5-S-2 dorsal and ventral
roots virtually abolished the increases in SP. This suggests that activation of fibers entering the spinal cord via
the L-5 and S-2 spinal roots elicits small increases in SP
release in the L-7 dorsal horn. Further, these results
suggest that movement of the lower back during a
muscle contraction is not responsible for the evoked
increases in SP release. In addition, the changes in SP
release are not the result of electrical activation of the
afferents, because changes in peptide release did not
occur when the contractions were prevented.
In the present study, the HT contractions were
evoked by stimulating the L-7 and S-1 ventral roots,
whereas only the L-7 was stimulated to elicit the LT
contractions. This was done because it is our experience
that the peak tension evoked by stimulating both roots is
more sustained than if the L-7 root is stimulated alone.
Because the design of the study was to compare HT and
LT contractions and because multiple contractions were
required, we wanted to ensure that the HT contractions
were reproducible and that the peak tension was sustained as long as possible. However, this raises the
possibility that the greater release of SP during the HT
contractions was the result of this neuropeptide being
released at different spinal levels. Since both ventral
roots innervate the triceps surae muscle, the afferents
activated by a contraction elicited by L-7 and S-1
stimulation should be the same as the ones activated by
L-7 stimulation alone (if equal tensions are produced).
Thus, it is very unlikely that the rostral-caudal extent of
SP release is different. Further, the probe was placed in
the center of the rostral-caudal extent of the L-7 dorsal
horn. Unfortunately, we do not know the rostral-caudal
extent from which the microdialysis probe samples. The
ability to determine this is a difficult problem, because
numerous factors (diffusion coefficients, rate of removal
from the extracellular space, size of the membrane, flow
rate, etc) influence the size of the sample area. Bungay
et a141 have suggested that the extent of delivery of
solutes to the extracellular space may be as great as 3
mm (depending on the aforementioned variables). The
L-7 region of the cat extends 7 to 11 mm, depending on
the size of the cat. Further, it should be kept in mind
that the L-5, L-6, and S-2 dorsal and ventral roots were
sectioned before inserting the microdialysis probe. Also,
the release of contraction-induced SP was abolished, as
were the cardiovascular changes, when the contractions
were repeated after cutting the L-7 and S-1 dorsal roots.
Thus, it is very unlikely that the SP measured in this
study was from an area outside the L-7 region.
In summary, HT contractions elicited greater increases in the release of SP in the L-7 dorsal horn
region of the spinal cord compared with LT contractions. This indicates that the release of SP is related to
the developed tension. Concomitant with the increased
SP release, HT contractions evoked larger cardiovascular changes. Thus, the results of the present study
suggest that the release of SP in the spinal cord plays a
role in mediating the cardiovascular responses produced by static contraction of skeletal muscle.
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.
The authors express their gratitude to James Jones and David
Maass for their expert technical assistance.
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Effects of graded muscle contractions on spinal cord substance P release, arterial blood
pressure, and heart rate.
L B Wilson, I E Fuchs and J H Mitchell
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Circ Res. 1993;73:1024-1031
doi: 10.1161/01.RES.73.6.1024
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