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Departments of Pharmacology, Biochemistry and Anatomy, University of New England College of Osteopathic Medicine, Biddeford, Maine 04005
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The effects of an opioid agonist, [D-Ala2]methionine enkephalinamide (DAME), administered into the rostral ventrolateral medulla (rVLM) or caudal ventrolateral medulla (cVLM) on cardiovascular responses to isometric muscle contraction were determined in anesthetized rats. A 30-s contraction evoked by tibial nerve stimulation increased mean arterial pressure (MAP) and heart rate (HR) by 34 ± 6 mmHg and 40 ± 7 beats/min, respectively, with a developed tension of 322 ± 30 g, after bilateral insertion of microdialysis probes into the rVLM. Thirty-minute dialysis of DAME (10 and 100 µM) attenuated the contraction-evoked cardiovascular changes dose dependently (10 µM: MAP = 25 ± 4 mmHg, HR = 27 ± 3 beats/min, tension = 333 ± 25 g; 100 µM: MAP = 14 ± 4 mmHg, HR = 16 ± 5 beats/min, tension = 330 ± 34 g). Preadministration of an opioid antagonist, naloxone (100 µM), augmented contraction-evoked MAP and HR responses and blocked effects of 100 µM DAME. Microdialysis of DAME into the cVLM produced no changes in the pressor response to contraction. At end of each experiment, tibial nerve stimulation after neuromuscular blockade evoked no MAP or HR change. Results demonstrate that opioid receptor activation within the rVLM modulates cardiovascular responses to isometric muscle contraction.
caudal ventrolateral medulla; arterial pressure; rat; naloxone
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ISOMETRIC MUSCLE contraction has been known to bring about cardiovascular changes in anesthetized cats (4, 6-8, 15, 19, 20), rats (2, 17), and chickens (31). Both heart rate (HR) and mean arterial pressure (MAP) increase in response to isometric muscle contraction and are commonly known as the exercise pressor reflex (20). As work is performed, impulses are transmitted via thinly myelinated (type III) and unmyelinated (type IV) muscle afferents to the neurons in the spinal cord and from there to higher centers in the brain (16, 19, 21). The neural information is then integrated in central cardiovascular regulatory areas. One region of particular importance is the ventrolateral medulla (VLM).
The VLM has been divided into two functional areas, the rostral VLM (rVLM) and the caudal VLM (cVLM), since chemical stimulation of the neurons in these areas evokes pressor and depressor responses, respectively (11, 37). The significance of the VLM as a regulatory area of cardiovascular function during static muscle contraction has been substantiated by previous studies (1, 7, 15). For instance, VLM neurons have been shown to be influenced by static exercise, in which an increase in discharge frequency by the neurons located in the VLM was observed during muscle contraction (7). Furthermore, a previous study has revealed that an increased metabolic rate occurs in the VLM during contraction (15). Thus the VLM appears to play a key role in cardiovascular function by integrating and regulating cardiovascular responses during isometric or static muscle contraction.
It is apparent that VLM neurons are under the influence of various neural mechanisms that modulate cardiovascular responses during muscle contraction. Cholinergic and excitatory amino acidergic neuromodulatory mechanisms in the VLM have been found to play a role in influencing the cardiovascular responses to static exercise in anesthetized cats (4, 6). Moreover, our recent study using anesthetized rats demonstrated a 5-hydroxytryptamine (5-HT; serotonergic) mechanism associated with the exercise pressor reflex, in which activation of 5-HT1A receptors in the rVLM but not in cVLM attenuates pressor and tachycardic responses to isometric muscle contraction (2). Another group of neuromodulators that has been shown to play an important functional role in regulating the cardiovascular system is endogenous opioids (14, 26). Opioid receptors have been located throughout the central nervous system (5, 14) and have been specifically localized in the VLM (13, 24, 25). Activation of opioid receptors within the rVLM causes a depressor effect (24), possibly mediated via a reduction in sympathetic nerve activity (23, 24). Furthermore, central opioid receptors play a role in the exercise pressor reflex, since administration of opioid agonists into the cerebral aqueduct of anesthetized cats attenuates the cardiovascular responses to static muscle contraction (39). The site of action of opioid receptor activation has been suggested to be the VLM (39, 40). However, the precise location of the opioid receptors, whether in the rVLM or the cVLM, has not been established. Therefore, the purpose of this study was to investigate the role and location of opioid receptors within the rVLM and cVLM involved in modulating cardiovascular responses during isometric muscle contraction.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Surgery
All protocols were approved by the Institutional Animal Care and Use Committee of the University of New England and conform with the rules and regulations established by the Animal Welfare Acts. Rats (Sprague-Dawley) of either sex weighing between 250 and 350 g were initially anesthetized by intraperitoneal administration of a combination of chloral hydrate (75 mg/kg) and pentobarbital sodium (25 mg/kg). The rats were monitored for corneal reflexes or a response to noxious stimuli to the paw throughout the experiment. If either was present, the animal was given an additional dose of chloral hydrate (75 mg/kg). Catheterization of a common carotid artery was performed to record arterial pressure (AP), MAP, and HR. These parameters were charted continuously on a Grass polygraph (model 79D, Grass Instruments, Quincy, MA). HR was triggered by the AP signal, and the MAP was integrated from the AP (time constant: 2 s). An external jugular vein and the trachea were also cannulated for intravenous injection of pancuronium bromide (200 µg/kg) and artificial respiration, respectively, for the experiments in which neuromuscular blockade was induced. The body temperature was monitored via a rectal probe and maintained between 37 and 38°C with a heating pad and an infrared heat lamp.Next, surgical preparation of the sciatic nerve and triceps surae muscle was performed as discussed in previous papers (2, 17). Briefly, the left sciatic nerve was exposed, and its tibial, peroneal, and sural branches were separated from each other. The tibial nerve was placed on a bipolar platinum electrode connected to a Grass stimulator (model S88) coupled to a Grass stimulus isolation unit (model SIU5C). The skin of the left triceps surae muscle was detached, and the muscle was isolated when the calcaneal bone was dissected. The Achilles tendon was affixed to a Grass force transducer (model FT03) for measurement of developed tension during periods of stimulation-induced muscle contraction. During the experiment, paraffin-saturated gauze kept the muscle and tendon moist. To prevent movement during periods of muscle contraction, the hips and the left knee joint were secured to metal clamps and a steel post, respectively.
Microdialysis
After the surgical setup was completed, the head of the rat was fixed in a stereotaxic frame. The teeth of the rat were secured with the bite bar set at 12 mm below the interaural line. Muscles located in the dorsal area of the neck were withdrawn to reveal the underlying occipital bone and the atlantooccipital membrane. Next, the occipital membrane was cut so that the dura over the cerebellum could be moved aside for exposure of the cerebellum. After that, the cerebellum was retracted so the fourth ventricle could be observed as far as the inferior cerebellar peduncle. Then a static muscle contraction was induced by stimulating the tibial nerve for 30 s at three times motor threshold, 0.1-ms duration, and a frequency of 40 Hz. AP, MAP, HR, and developed tension were measured during the contraction period. This contraction served as a control to determine whether a later contraction (see below) after bilateral insertion of microdialysis probes inflicted tissue destruction rendering the area functionally damaged. Subsequently, two microdialysis probes, each with a diameter of 0.24 mm and 1-mm membrane tip (CMA-11, CMA/Microdialysis, Acton, MA), were inserted bilaterally into either the rVLM or cVLM. Coordinates for probe insertions were determined using the caudalmost point of the area postrema as zero reference. The rVLM coordinates were established as 2 mm rostral to the caudal tip of area postrema, 1.9 mm lateral, and 2.4 mm ventral to the floor of the fourth ventricle (27). For the cVLM, probes were inserted 0.5 mm rostral to the caudal tip of the area postrema (27). The probes were perfused continuously with artificial cerebrospinal fluid (CSF) at a rate of 0.5 µl/min. The artificial CSF was composed of 125 mM NaCl, 1.26 mM CaCl2, 2.5 mM KCl, and 1.18 mM MgCl2 in sterile water. All drugs were dissolved in this solution. The pH and osmolality were adjusted to 7.4 and ~309 mosmol/kg, respectively.Experimental Protocols
L-Glutamate (10 nM) was microdialyzed bilaterally at the beginning of each experiment to functionally assess whether the probes' membranes were within the rVLM or cVLM. Pressor and depressor responses confirmed the position of the probes within the rVLM or cVLM, respectively.Protocol I. Effects of metenkephalinamide microdialysis into rVLM. Once the location of the probes was confirmed to be within the rVLM by L-glutamate, a 120-min stabilization period ensued. During that time, artificial CSF was constantly perfused. At the completion of the stabilization period, the second static muscle contraction was evoked. A 30-s tibial nerve stimulation (3 × motor threshold, 0.1 ms, 40 Hz) was performed to contract the left triceps surae muscle of the rat. Baseline and contraction-induced changes in AP, MAP, HR, and tension were recorded. After the contraction, [D-Ala2]methionine enkephalinamide (DAME; Sigma Chemical, St. Louis, MO) was microdialyzed first at 10 µM concentration followed by 100 µM (n = 5). A static muscle contraction was performed using the above-mentioned parameters after 30 min of perfusing each dose. Further experiments were executed using 100 µM DAME, since this dose was determined to be most effective (n = 10). After a muscle contraction subsequent to 100 µM DAME, the drug perfusion was discontinued, and artificial CSF was dialyzed through the probes for an additional 60 min to determine whether the cardiovascular responses to muscle contraction returned to predrug values (recovery). At the completion of each experiment, the rats were given an injection of pancuronium bromide (200 µg/kg iv) to induce paralysis. The rats were ventilated artificially with a Harvard respirator (model 681) in room air, 60 strokes/min, 1 ml/100 g body wt. Thereafter, the tibial nerve was stimulated using the prior stimulation parameters. The purpose of paralyzing the animals and subsequent tibial nerve stimulation was to demonstrate that the type III and IV muscle afferents were not being activated directly by the electrical stimulation (see Ref. 2 for representative tracing).
EFFECTS OF DAME MICRODIALYSIS INTO CVLM. After the the probes were placed within the cVLM, a 120-min stabilization period was allowed while artificial CSF was perfused. The tibial nerve was then stimulated for 30 s as described above to evoke a muscle contraction, and AP, MAP, HR, and tension were recorded. After the contraction, 100 µM DAME was microdialyzed for 30 min into the cVLM (n = 5). Another muscle contraction was repeated using the parameters mentioned above. Finally, the tibial nerve was stimulated after the animal was paralyzed with pancuronium bromide as described above.Protocol II. Effects of naloxone microdialysis into rVLM and cVLM. The effects of an opioid receptor antagonist, naloxone (100 µM, Sigma), on the MAP and HR responses to tibial nerve stimulation-evoked muscle contraction were determined by microdialyzing the drug into the rVLM for 30 min. MAP and HR were recorded before and during the contraction. Then 100 µM DAME was microdialyzed for 30 min, another static muscle contraction was evoked, and MAP and HR were measured. Finally, a tibial nerve stimulation was performed after neuromuscular blockade. In five anesthetized rats, 100 µM naloxone was microdialyzed into the cVLM after a muscle contraction. A contraction was repeated after 30 min, after which 100 µM DAME was perfused for an additional 30 min. Thereafter, another muscle contraction was elicited while cardiovascular responses were measured.
Histology
At the end of each experiment, we established the location of the microdialysis probes and estimated the distribution of the drugs microdialyzed into the rVLM and cVLM by perfusing the probes with a solution of 100 µM methylene blue for 30 min, the time corresponding to the duration of drug delivery. The animal was then perfused transcardially with 0.9% saline and thereafter with 10% phosphate-buffered formaldehyde solution. The brain was removed, placed in a 10% phosphate-buffered formaldehyde solution, and stored at 4°C until histology was performed. Transverse sections (50 µm) through the medulla were cut using a Vibratome, and anatomic verification of probe placement and the rostrocaudal distribution of the dye were visualized with a microprojector in all the animals.Statistical Analyses
Data (AP, MAP, HR, and tension) were recorded on a four-channel chart recorder (Grass polygraph model 79D). Baseline values were calculated from the data collected for at least 2 min before each contraction. Peak values for each variable were the maximal response evoked during the 30-s contraction period. Data are expressed as means ± SE. All data were initially tested for normality, and because the test passed, parametric statistical analyses were carried out. Dose-response effect of DAME on MAP and HR changes was analyzed using a one-way analysis of variance with repeated measures (RMANOVA). Baseline and peak hemodynamic variables during muscle contractions before and after placement of probes and after 100 µM DAME dialysis were compared using the one-way RMANOVA. Statistical analyses of cardiovascular and tension data before and after naloxone and after DAME were assessed by the one-way RMANOVA. Tukey's test was used for all post hoc analyses. The significance level for all analyses was taken as P < 0.05.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cardiovascular Responses Before and After Probe Insertion
For all experiments before the probes were introduced into the rVLM (n = 20), MAP and HR were measured and recorded during a static muscle contraction. MAP increased after the tibial nerve stimulation-evoked contraction from 105 ± 4 to 135 ± 5 mmHg (
MAP = 30 ± 4 mmHg). Similarly, HR increased from
400 ± 6 to 437 ± 5 beats/min (HR = 37 ± 5 beats/min).
Bilateral insertion of the probes did not significantly affect the
contraction-induced changes in MAP or HR, since MAP increased by 32 ± 5 mmHg (108 ± 4 to 140 ± 5 mmHg) and HR by 35 ± 5 beats/min (410 ± 6 to 445 ± 7 beats/min). Developed
tensions were also similar: 350 ± 20 vs. 360 ± 25 g, pre- vs.
post-probe insertion, respectively.
L-Glutamate (10 nM) was then
microdialyzed bilaterally into the rVLM. An immediate pressor response
of 30 ± 3 mmHg and an increase in HR of 40 ± 4 beats/min were
attained after L-glutamate. The
increases in HR and MAP demonstrated that the probe membranes were
located within the rVLM.
Before the probes were inserted into the cVLM for all experiments
(n = 10), tibial nerve
stimulation-evoked muscle contraction increased MAP from 110 ± 5 to
142 ± 4 mmHg (MAP = 32 ± 4 mmHg) and HR from 400 ± 8 to 440 ± 7 beats/min (HR = 40 ± 7 beats/min). After
bilateral insertion of the probes, tibial nerve stimulation increased
similar MAP and HR changes of 30 ± 5 mmHg and 39 ± 4 beats/min,
respectively. Developed muscle tensions were also similar. Next,
L-glutamate administration
confirmed the placement of the probes within the cVLM by eliciting a
depressor response of 35 ± 4 mmHg and bradycardia of 
34 ± 3 beats/min.
Dose-Dependent Effects of DAME Microdialysis Into rVLM
In this series of experiments, tibial nerve stimulation after bilateral probe insertion into the rVLM (control) evoked cardiovascular responses with a developed tension of 322 ± 30 g (n = 5; Fig. 1). MAP and HR increased by 34 ± 6 mmHg and 40 ± 7 beats/min, respectively (n = 5; Fig. 1). Bilateral microdialysis of DAME at doses of 10 µM followed by 100 µM, each for 30 min, into the rVLM affected the pressor and HR responses to subsequent muscle contraction in a dose-dependent manner without a change in baseline cardiovascular variables (Fig. 1). Developed tensions during each contraction period were similar throughout the protocol. The 100 µM dose of DAME was used in a set of additional experiments, since it attenuated cardiovascular responses generated during the muscle contractions more than those after the lower dose of 10 µM.
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Effects of 100 µM DAME Microdialysis Into rVLM
Before microdialysis of 100 µM DAME into the rVLM (n = 10), a tibial nerve stimulation-evoked muscle contraction increased MAP from a basal level of 104 ± 5 mmHg to a peak level of 133 ± 6 mmHg (P < 0.05), i.e., a change of 29 ± 3 mmHg (Fig. 2). Similarly, HR was also elevated during the 30-s isometric contraction period. HR increased by 37 ± 4 beats/min (Fig. 2): the basal and peak absolute values were 420 ± 20 and 457 ± 18 beats/min (P < 0.05), respectively. These cardiovascular changes in response to static muscle contractions were significantly attenuated after the microdialysis of DAME for 30 min without any shift in baseline MAP or HR (Fig. 2).
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Blood pressure response to the contraction was 12 ± 3 mmHg
(baseline and peak absolute values were 107 ± 5 and 119 ± 4 mmHg, respectively), whereas HR increased by 12 ± 4 beats/min (baseline and peak absolute values being 425 ± 10 and 437 ± 8 beats/min, respectively). Muscle tensions during the
contractions before and after DAME were similar (Fig. 2). These data
represent a 70% attenuation of the cardiovascular responses to
isometric muscle contraction after administration of an opioid agonist
into the rVLM. After the muscle contraction, perfusion of DAME was
discontinued, and artificial CSF was dialyzed through the probes for 60 min. A muscle contraction was evoked, and cardiovascular responses recovered to predrug values (MAP = 27 ± 4 mmHg and HR = 34 ± 4 beats/min), suggesting that the functional integrity of the area is intact (Fig. 2).
Effects of 100 µM DAME Microdialysis Into cVLM
In five anesthetized rats before microdialysis of 100 µM DAME into the cVLM, muscle contraction increased MAP from 100 ± 5 to 135 ± 5 mmHg (MAP = 35 ± 4 mmHg;
P < 0.05), and HR increased from 395 ± 10 to 425 ± 9 beats/min (HR = 30 ± 4 beats/min;
P < 0.05). Microdialysis of DAME for
30 min had no effect on baseline MAP or HR or cardiovascular responses
during muscle contractions. Blood pressure increased from 98 ± 5 to
135 ± 4 mmHg (MAP = 37 ± 4 mmHg). Similarly, HR increased by
29 ± 4 beats/min, i.e., from 400 ± 9 to 429 ± 8 beats/min.
The developed tensions before and after DAME were 430 ± 20 and 440 ± 25 g, respectively. These data demonstrate that administration of
the opioid agonist into the cVLM had no effect on cardiovascular
responses elicited during isometric muscle contraction.
Effects of 100 µM Naloxone Microdialysis Into rVLM and cVLM
The opioid receptor antagonist naloxone (100 µM) was microdialyzed into the rVLM for 30 min. Naloxone did not alter baseline MAP or HR (prenaloxone: MAP = 100 ± 4 mmHg, HR = 410 ± 7 beats/min; postnaloxone: MAP = 104 ± 5 mmHg, HR = 406 ± 7 beats/min). However, administration of naloxone potentiated the cardiovascular responses during subsequent static muscle contraction (n = 5; Fig. 3). An increase of 40 ± 4 mmHg in MAP was exhibited after naloxone vs. 27 ± 3 mmHg (P < 0.05) before the drug (Fig. 3). Likewise, the HR response during static contraction was potentiated after naloxone, i.e., a change of 49 ± 4 beats/min after naloxone vs. 36 ± 3 beats/min before the drug (P < 0.05). The tibial nerve stimulation-evoked developed tensions were 490 ± 27 and 500 ± 20 g before and after naloxone, respectively. However, naloxone abolished the attenuating effects of subsequent 30-min microdialysis of 100 µM DAME (Fig. 3). Administration of naloxone into the cVLM (n = 5) had no effects on baseline MAP or HR or on cardiovascular changes in response to muscle contraction. Furthermore, muscle contraction after a subsequent 30-min microdialysis of DAME produced no changes in MAP and HR responses (MAP, mmHg:
prenaloxone 24 ± 3, postnaloxone 25 ± 5, post-DAME 23 ± 3;
HR, beats/min: prenaloxone 30 ± 4, postnaloxone 34 ± 6, post-DAME 33 ± 3). These results of naloxone administration into
the cVLM corresponds to those of a previous study in which localized
injection of the drug into the cVLM had no effects on HR or blood
pressure (38).
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Effects of Tibial Nerve Stimulation After Neuromuscular Blockade
At the end of each experiment, the rats were paralyzed by an intravenous administration of pancuronium bromide (200 µg/kg) and were artificially ventilated using a respirator. The tibial nerve was then stimulated using prior parameters. No changes in MAP or HR were observed, suggesting that the cardiovascular responses were due to contraction-induced activation of muscle afferents (see Refs. 1 and 2 for representative tracings).Histological Analysis of Dye Distribution
Sections of the brain established that the membranes of the probes were in the rVLM and the cVLM and matched the planes in the rat brain atlas (27). Furthermore, visualization of histological sections estimated the spread of methylene blue dye for 30 min in the region. Around each microdialysis probe, the average dye diffusion into the surrounding tissues was ~600 µm (Fig. 4). This diffusion was observed in all experiments and corresponds to the pattern of distribution shown by recent studies using similar techniques (2, 17).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We examined the effects of bilaterally infusing DAME, an opioid receptor agonist, into the rVLM and cVLM on cardiovascular responses elicited during static muscle contraction using anesthetized rats. The results demonstrated an attenuation of the increases in MAP and HR during tibial nerve stimulation-evoked muscle contraction after microdialysis of DAME into the rVLM but not into the cVLM. Our findings point to a rVLM-mediated opioidergic mechanism that plays a role in mediating cardiovascular responses to static muscle contraction.
Increases in HR and MAP in response to muscle contraction in
anesthetized cats, rabbits, and dogs have been documented by numerous
investigators (4, 6-8, 15, 16, 19, 20). In anesthetized rats,
demonstration of an exercise pressor reflex had been controversial
until recently, when two studies have been able to confirm and
establish that stimulation of the tibial nerve in the anesthetized rat
evokes a static muscle contraction associated with consistent increases
in MAP and HR (2, 17). It has been shown that tibial nerve stimulation
is associated with a fall in MAP in anesthetized paralyzed rats (32).
It is likely that paralyzing the rat may have caused the depressor
response (32), and furthermore, the above study (32) used stimulation
parameters 30 times motor threshold and ~200 times greater than those
required to detect a group III fiber response, suggesting direct
activation of muscle afferents. We demonstrated an elevation in MAP and
HR in response to a tibial nerve stimulation-evoked static muscle contraction in anesthetized unparalyzed rats when parameters similar to
those for anesthetized cats were used (4, 6-8, 19, 20, 39, 40).
These cardiovascular changes were mediated by contraction-induced activation of the type III (thinly myelinated) and IV (unmyelinated) muscle afferents, since at the end of each experiment after
neuromuscular blockade, stimulation of the tibial nerve had no effects
on MAP or HR. Furthermore, a recent study produced tetanic contractions in pentobarbital sodium-anesthetized rats using one times motor threshold as the stimulation parameter and showed a small, 10-mmHg increase in MAP (33). It is possible that the type of anesthetic used
and the stimulation parameter may have contributed to small rise in
blood pressure. Again, in another study using an
-chloralose-anesthetized Wistar rat preparation, a static muscle
contraction, evoked by stimulation of the tibial nerve, increased MAP
by 5 mmHg (34). It appears that the type of anesthetic, strain of rat,
and stimulation parameter of two times motor threshold had been
contributing factors in the small rise in blood pressure (34). It is of
note that static muscular contraction also elicits an exercise pressor
reflex in the anesthetized chicken (31).
The central integrator of the exercise pressor reflex has been
suggested to be the VLM (1, 6-8, 15). Although the VLM has been
suggested as a integration center of cardiovascular responses during
muscle contraction, it is also influenced by numerous neural inputs.
Activation of 5-HT1A receptors in
the rVLM, but not within the cVLM, reduces the changes in MAP and HR to
muscle contraction, demonstrating that a
5-HT1A mechanism plays a role in
mediating cardiovascular responses during static muscle contraction
(2). In addition, centrally located
2-adrenoceptors, muscarinic
cholinergic receptors, and excitatory amino acid receptors are involved
in modulating the pressor response during static exercise. When these receptors are stimulated or blocked by their respective agonists or
antagonists, cardiovascular changes to static muscular contraction are
attenuated (3, 4, 6, 39). A summary of the central nervous system
circuitry regulating cardiovascular responses during static muscle
contraction has been described extensively in a recent review (1).
Exercise and other stressful stimuli have been shown to be associated with an increase in endogenous opiates as measured from blood samples (10, 12). Also, endogenous opioids have been shown to influence the central cardiovascular control systems (14, 26). It has previously been demonstrated that the site of origin of opioidergic pathway projecting to the VLM is the paraventricular nucleus of the hypothalamus (30). Furthermore, presence of opioid receptors has been demonstrated in both the rVLM and cVLM, suggesting a role for enkephalins in cardiovascular regulation (5, 13, 14). Central administration of opioid agonists in anesthetized animals decreases HR and blood pressure (14, 26). Stimulation of the opioid receptors in the rVLM has been demonstrated to reduce MAP and HR (24, 28, 29). In contrast, MAP and HR increase after stimulation of opioid receptors located in the cVLM (28, 38). Furthermore, a previous study has shown that administration of 1-100 µg DAME into the cerebral aqueduct of anesthetized cats attenuates the pressor response to fatiguing muscle contraction without a change in baseline cardiovascular variables (39). However, when a drug is injected into the cerebral aqueduct of animals, it acts on wide nonspecific sites. In the present study, the opioid agonist DAME was microdialyzed directly into the rVLM and cVLM. Contraction-induced rises in MAP and HR were attenuated by local administration of DAME into the rVLM, but not when the drug was locally dialyzed into the cVLM. The present study extends the previous observation (39) on opioidergic modulation of the exercise pressor reflex by demonstrating that opioid receptors located in the rVLM, but not in the cVLM, play a role in mediating cardiovascular responses to muscle contraction. We demonstrated a site-specific rVLM-mediated opioidergic mechanism associated with the exercise pressor reflex.
Baseline values of MAP and HR were not altered by administration of DAME either into the rVLM or the cVLM. Previous investigations have shown that bilateral microinjection of DAME at a dose of 50 ng/site induces a significant decrease in baseline MAP and HR, and an increase when administered into the rVLM and cVLM, respectively, that lasts for 2-4 min (28, 29). In addition, for a long lasting effect (20-30 min) on the baseline MAP and HR, the dose of DAME was 500 ng/site (28, 29). The reason for the lack of change in baseline variables in our study can be attributed to the amount and long duration of DAME perfused in our experiments vs. that employed in the above studies (28, 29). We also performed percent recovery of DAME for microdialysis probes using in vitro tests. The amount of drug diffusion from the perfusing medium to solutions of known concentrations of DAME revealed a recovery rate of ~10%. It appears that the dose of DAME that diffused in the rVLM and cVLM was less (~8.6 ng/site) than the previous experiments where baseline MAP changed (28, 29). However, because the area immediately surrounding the microdialysis membrane in vivo is very dynamic during drug delivery, the actual concentration of DAME cannot be ascertained by the present study. Regardless, DAME had an attenuating effect on the pressor response to static muscle contraction.
Microdialysis of naloxone, an opioid receptor antagonist, into the rVLM blocked the attenuating effects of DAME, suggesting that DAME stimulated the opioid receptors in the rVLM. After naloxone was administered into the rVLM, static muscle contraction significantly potentiated the MAP and HR responses (see Fig. 3). This result indicates that rVLM opioid receptors that are blocked by the 100 µM dose of naloxone have a tonic influence on neurons mediating HR and blood pressure responses during static muscular contraction. Our results relate to a previous report in which the pressor response induced by stimulation of afferent nerves was potentiated (22). Although other reflex responses are increased after naloxone administration (9, 18), two studies have reported that intravenous administration of naloxone in either the anesthetized or the conscious cat had no effects on the pressor response during static exercise (35, 36). A possible reason for this difference is the different route of administration of naloxone or a species difference.
To demonstrate the site-specific effect of the drugs,
L-glutamate was used as a
control to determine proper placement of the membranes of the probes in
the rVLM or cVLM (see RESULTS).
Furthermore, at the completion of each experiment, methylene blue was
microdialyzed into each region for 30 min, and the diffusion of the dye
around the probe was found to be 
600 µm in all preparations. The
rostral or caudal VLM dimensions in the rat are ~1 × 1 × 1.5 mm (some 1.5 mm3). The
distribution of a dye cannot rule out the possibility of a wider spread
of the drugs to structures other than the rVLM or cVLM. However, this
seems unlikely, since microdialyzing DAME into the rVLM had an
attenuating effect on the increases in MAP and HR during the
contraction, and, in contrast, administration of the drug into the cVLM
(an area 1.5 mm caudal to rVLM) did not. If the drug had diffused out
of the cVLM, i.e., >500-600 µm, then an rVLM-mediated
attenuation would probably have occurred. Because this did not happen,
it was presumed that the effects of the drugs were localized and site
specific.
In conclusion, we demonstrated that the cardiovascular responses during static muscle contraction were inhibited after stimulation of opioid receptors located in the rVLM but not within the cVLM. In addition, our data showed that the opioid receptors in the rVLM that are blocked by the dose of naloxone used in the study exert a tonic influence on the neurons mediating HR and blood pressure responses during static muscle contraction.
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ACKNOWLEDGEMENTS |
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D. Caringi was supported by a fellowship from the American Heart Association (Maine Affiliate) and the University of New England Dean's Summer Research Grant.
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FOOTNOTES |
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Components of this study have been presented in abstract form (Soc. Neurosci. Abstr. 2: 852, 1996).
Address for reprint requests: A. Ally, Depts. of Pharmacology and Biochemistry, University of New England College of Medicine, 11 Hills Beach Rd., Biddeford, ME 04005.
Received 18 July 1997; accepted in final form 8 September 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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