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Abstract
Abstract Using α-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.
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
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 α-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
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.
Contraction-induced changes in MAP after each of three doses of DAME had been dialyzed sequentially into the L-7 dorsal horn for 40 minutes (n=3). *P<.05 compared with control (dose, 0 μmol/L). Values are mean±SEM.
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.
A representative tracing from one cat showing the pressor and tachycardiac responses and the developed tension to a 1-minute static muscle contraction of the triceps surae muscle before (left) and after (right) 80 minutes of 200 μmol/L DAME microdialysis into the L-7 dorsal horn.
Changes in MAP and HR and the tension developed during static muscle contraction of the triceps surae muscle. Drug1 indicates contraction after 49±4 minutes of DAME (n=9); drug2, contraction after 92±3 minutes of DAME (n=10). *P<.05 vs control (n=10) and drug1; +P<.05 vs drug2. Values are mean±SEM.
Hemodynamic and Tension Data in Response to Muscle Contraction and Passive Stretch Before and After 200 μmol/L DAME
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.
A representative tracing from one cat showing the pressor and tachycardiac responses to a 1-minute passive stretch of the triceps surae muscle before (left) and after (right) 80 minutes of 200 μmol/L DAME microdialysis.
Changes in MAP and HR and developed tension during stretch of the triceps surae muscle. Drug1 indicates contraction after 52±3 minutes of DAME (n=9); drug2, contraction after 97±5 minutes of DAME (n=10). *P<.05 vs control (n=10) and drug1. Values are mean±SEM.
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⇓).
Left, Mean peak changes in MAP and HR and developed tension during the 5-minute static contractions before and after 200 μmol/L DAME microdialysis while the dialysate was collected for measurement of SP-LI (n=8). Right, Corresponding data for the time controls, for which no drug was dialyzed (n=2). DAME indicates contraction after drug dialysis; contract 1 and 2, first and second contractions, respectively, for the time control. Values are mean±SEM.
Left, Changes in SP-LI before (control) and after DAME microdialysis (n=8). Right, Changes in SP-LI during the two contractions of the time control experiments (n=2). DAME indicates contraction after drug dialysis; contract 1 and 2, first and second contractions, respectively, for the time control. Values are mean±SEM.
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
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 α2-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
| AP | = | arterial pressure |
| DAME | = | [d-Ala2]-methionine enkephalinamide |
| ECF | = | extracellular fluid |
| HR | = | heart rate |
| MAP | = | mean arterial pressure |
| MT | = | motor threshold |
| RIA | = | radioimmunoassay |
| RM-ANOVA | = | repeated-measures ANOVA |
| SP | = | substance P |
| SP-LI | = | SP-like immunoreactivity |
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.
- © 1995 American Heart Association, Inc.
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