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Am J Physiol Heart Circ Physiol 287: H1312-H1318, 2004. First published April 15, 2004; doi:10.1152/ajpheart.00163.2004
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Central integration of muscle reflex and arterial baroreflex in midbrain periaqueductal gray: roles of GABA and NO

Jianhua Li

Division of Cardiology, Department of Medicine, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; andInternal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75235

Submitted 17 February 2004 ; accepted in final form 9 April 2004


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It has been suggested that the midbrain periaqueductal gray (PAG) is a neural integrating site for the interaction between the muscle pressor reflex and the arterial baroreceptor reflex. The underlying mechanisms are poorly understood. The purpose of this study was to examine the roles of GABA and nitric oxide (NO) in modulating the PAG integration of both reflexes. To activate muscle afferents, static contraction of the triceps surae muscle was evoked by electrical stimulation of the L7 and S1 ventral roots of 18 anesthetized cats. In the first group of experiments (n = 6), the pressor response to muscle contraction was attenuated by bilateral microinjection of muscimol (a GABA receptor agonist) into the lateral PAG [change in mean arterial pressure ({Delta}MAP) = 24 ± 5 vs. 46 ± 8 mmHg in control]. Conversely, the pressor response was significantly augmented by 0.1 mM bicuculline, a GABAA receptor antagonist ({Delta}MAP = 65 ± 10 mmHg). In addition, the effect of GABAA receptor blockade on the reflex response was significantly blunted after sinoaortic denervation and vagotomy (n = 4). In the second group of experiments (n = 8), the pressor response to contraction was significantly attenuated by microinjection of L-arginine into the lateral PAG ({Delta}MAP = 26 ± 4 mmHg after L-arginine injection vs. 45 ± 7 mmHg in control). The effect of NO attenuation was antagonized by bicuculline and was reduced after denervation. These data demonstrate that GABA and NO within the PAG modulate the pressor response to muscle contraction and that NO attenuation of the muscle pressor reflex is mediated via arterial baroreflex-engaged GABA increase. The results suggest that the PAG plays an important role in modulating cardiovascular responses when muscle afferents are activated.

blood pressure; exercise pressor reflex

THREE MECHANISMS ARE THOUGHT to contribute to autonomic responses to exercise. First, afferent input arising from contracting skeletal muscle relayed to the brain stem increases cardiovascular activities (5, 26, 31, 32). Mechanically and metabolically sensitive group III and group IV afferent nerve endings (receptors) located in the muscle are engaged by contraction and initiate a reflex (5, 16, 17, 26). The reflex cardiovascular responses to muscle contraction are termed the "exercise pressor reflex" (26, 31, 32). The second mechanism, termed "central command" (11, 44), suggests that, during exercise, afferent input to the brain stem that elicits cardiovascular responses originates in higher centers of the brain, such as the motor cortex and/or subcortical motor areas (10, 45). The third source of inputs to the brain stem during exercise arises from peripheral baroreceptor populations. While neural signals from the exercise pressor reflex and central command are activated during exercise, inputs from arterial baroreceptors are constantly being transmitted to the central nervous system. It is thought that the arterial baroreflex modulates cardiovascular responses due to engagement of the exercise pressor reflex and central command (39–41, 46). The neural mechanisms by which the arterial baroreflex alters the exercise pressor reflex remain unclear.

The midbrain periaqueductal gray (PAG) is an important neural substrate for autonomic regulation (1, 24, 42, 43) and plays a role in regulating the arterial baroreflex (14, 34, 43). The PAG is also linked to the exercise pressor reflex (15, 19, 47, 48). For example, muscle contraction increases PAG neuropeptide Y and enkephalin release in cats (47, 48). In addition, treadmill exercise in rats activates PAG neurons (15), and muscle contraction increases PAG neuron discharge (19).

The studies further suggest that the PAG is a neural integrating site for the interaction between the exercise pressor reflex and the arterial baroreflex. In a previous study, c-Fos expression was used to identify activated neuronal cells (22). The results of this report suggested that skeletal muscle and baroreceptor afferent inputs activated neuronal cells in the PAG during muscle contraction. Second, direct neuronal projections from the dorsal horn of the spinal cord, the first synaptic site of the exercise pressor reflex, terminate in the PAG neurons (6, 18) that are activated by the arterial baroreflex inputs (22, 33). Third, convergence of afferent inputs from muscle receptors and arterial baroreceptors in the PAG inhibits the release of the excitatory amino acid glutamate (21).

The purpose of this study was to examine the roles of {gamma}-aminobutyric acid (GABA) and nitric oxide (NO) within the PAG in modulating the exercise pressor reflex. The first hypothesis that an increase in GABA within the PAG attenuates the exercise pressor reflex was tested. NO has been reported to be involved in the cardiovascular regulation in the PAG (7, 12). The effect of NO in the PAG on the cardiovascular responses is mediated in part by increased presynaptic vesicular release of GABA (12). Furthermore, it has been shown that neuronal processes containing NO synthase have close contacts with muscle contraction-activated neurons in the PAG (20). Thus the second hypothesis was that an increase in NO formation attenuates the exercise pressor reflex via GABA mechanisms in the PAG. The results from this study support these hypotheses.


   METHODS
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 ABSTRACT
 METHODS
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General procedures. All experimental procedures were approved by the Animal Care Committee of the Pennsylvania State University College of Medicine and complied with National Institutes of Health guidelines for the care and use of laboratory animals.

Anesthetized cats (n = 18) of either gender (4.3–5.8 kg body wt) were anesthetized initially with ketamine (25 mg/kg im) and then by inhalation of 2–5% isoflurane in 100% O2. A tube was inserted into the trachea via a tracheotomy to maintain an open airway, and a femoral vein and artery were cannulated for drug administration and measurement of arterial blood pressure, respectively. The gaseous anesthetic was discontinued after {alpha}-chloralose (80 mg/kg) and urethane (200 mg/kg) were injected intravenously. Throughout the experiment, supplemental injections of {alpha}-chloralose (15 mg/kg) and urethane (40 mg/kg) were given if the cats exhibited a corneal reflex or withdrew a limb in response to a noxious stimulus. The respiratory activities were monitored a pneumotachograph (Fleisch) connected to a respiratory gas monitor (Datex-Ohmeda, Madison, WI). Arterial blood gases and pH were also periodically checked (RapidLab 865 blood gas analyzer, Bayer) and maintained within normal limits (pH 7.30–7.40, 32–36 mmHg PCO2, >80 mmHg PO2) by adjustment of the ventilator (model 661, Harvard Apparatus, South Natick, MA) or intravenous injection of 1 M sodium bicarbonate. Body temperature was continuously monitored with a rectal probe and maintained at 37.5–38.5°C by a water-perfused heating pad and an external heating lamp.

Additional surgery was performed to allow acute sinoaortic and cardiopulmonary baroreceptor deafferentation (21, 37). The ventral surface of the neck was exposed by a midline incision. Superficial tissues were cauterized to expose the common carotid arteries and the carotid sinus bifurcation bilaterally. Silk sutures were then placed around the vagosympathetic nerves bilaterally to isolate input from aortic and cardiopulmonary baroreceptors. Sutures were also placed bilaterally around the carotid sinus nerve, the internal carotid artery, and the occipital artery distal to the carotid sinus bifurcation.

Arterial blood pressure was measured with a pressure transducer (model P23ID, Statham, Oxnard, CA) connected to an arterial catheter. Mean arterial pressure (MAP) was obtained by integrating the arterial signal with a time constant of 4 s. Heart rate (HR) was derived from the arterial pressure pulse. All measured variables were continuously recorded on an eight-channel chart recorder (model TA 4000, Gould Instruments, Valley View, OH). These variables were also sampled by an iMac computer that was equipped with PowerLab data acquisition system (ADInstruments, Castle Hill, Australia).

Laminectomy and muscle contraction. A laminectomy was performed to expose the lower lumbar and upper sacral portions of the spinal cord. The dura was then opened. The L7 and S1 spinal ventral roots were carefully separated and cut close to the spinal cord. The peripheral ends of the transected L7 and S1 ventral roots were then placed on platinum bipolar stimulating electrodes, and the exposed spinal cord region was immersed in a pool of warm (37°C) mineral oil. The calcaneal bone of one hindlimb was cut, allowing the Achilles tendon to be connected to a force transducer for measurement of developed tension during electrically stimulated muscle contraction. The pelvis was stabilized in a spinal unit (Kopf Instruments, Tujunga, CA), and the knee joints were secured by attachment of the patellar tendon to a steel post. Muscle contraction was induced by electrical stimulation of the L7 and S1 ventral roots (3 times motor threshold, 0.1-ms duration, 40 Hz).

Craniotomy and microinjection. The cat's head was fixed in a stereotaxic apparatus (Kopf Instruments), and a craniotomy was performed to expose the brain stem and cerebellum. For placement of micropipettes into the PAG, the superior colliculi were exposed by gentle reflection of the cortex, removal of a portion of the cerebellum by suction, and removal of a portion of the tentorium. Gelfoam was used to minimize any bleeding during this procedure, and warmed (37°C) mineral oil was applied to the dorsal surface of the superior colliculi.

The glass micropipette (30 µm tip diameter) was connected to a Nanoinject II system (Drummond Scientific) for delivery of each injection. A stereotaxic carrier (Kopf Instrument) was used to position the micropipette into the middle PAG at A0.6 (middle PAG) (2). Each micropipette was inserted ~2.0 mm below the dorsal surface of the brain stem and 1.2 mm lateral to midline to reach the lateral regions of the PAG. It has been reported that this area was activated by static muscle contraction (22). Local microinjection was performed via a Nanoinject II system. The volume of microinjection was 0.1 µl. Functional confirmation of the accurate placement of micropipettes into these regions was verified by local delivery of 0.1 µl of 10 nM L-glutamate into the lateral regions of the PAG to produce an immediate increase in arterial blood pressure.

Muscimol, bicuculline, L-arginine, and N-nitro-L-arginine methyl ester (L-NAME) were dissolved in an artificial extracellular fluid, which consisted of (per 100 ml of distilled H2O) 37.3 mg KCl, 724.9 mg NaCl, 15.7 mg MgSO4, 109.2 mg NaHCO3, 16.9 mg KH2PO4, 200 mg bacitracin, 200 mg bovine albumin, and 35.3 mg CaCl2·H2O and was filtered, degassed, and buffered to pH 7.35–7.40 with 0.1 N NaOH and HCl.

Experimental protocol. After the surgical procedures, 90 min were allowed for stabilization of the preparation. In the first group of studies (n = 6), the effects of the GABA agonist muscimol (0.01, 0.1, and 1 mM) on the exercise pressor reflex were determined. After placement of the glass micropipette, a 30-min "rest" period was employed so that basal levels of MAP and HR could be achieved. Next, a 1-min period of muscle contraction was performed as a control. This was followed by a 30-min recovery period. The first dose of muscimol was then injected into the PAG, and the muscle was contracted for 1 min. Sequential doses of muscimol were then injected. Each injection was followed by 1 min of muscle contraction and 30 min of recovery. Each contraction was performed 10 min after the injections. In this experiment, 0.01 mM bicuculline was also injected before muscimol to determine whether the effect of GABA activation was selective. In four animals, the effect of 0.01 and 0.1 mM bicuculline on the exercise pressor reflex was also examined. In these animals, the effect of GABA blockade was further determined after sinoaortic denervation and vagotomy.

In another group of studies (n = 8), basal levels of MAP and HR were obtained 30 min after placement of the glass micropipette. The muscle was then contracted for 1 min and allowed to recover for 30 min. Then, 5 mM L-arginine was injected, and 10 min later, the muscle was contracted for 1 min. The effects of L-arginine were also examined after microinjection of 10 mM L-NAME. Whether the effects of NO were mediated via GABA was also determined by injection of the GABAA receptor antagonist bicuculline (0.01 mM) before L-arginine. This dose of bicuculline did not affect the pressor response observed during muscle contraction. This afforded the opportunity to precisely determine the effect of NO on the reflex muscle response. Finally, the effects of the denervation on NO attenuation were examined (n = 6). In this study, the precursor of NO synthesis, L-arginine, was injected. This approach was based on a previous report (25) demonstrating that L-arginine and an NO donor have the same effects on the NO-mediated neuronal activity in the brain stem.

Afferent activity from aortic and cardiopulmonary baroreceptors was eliminated after bilateral transection of the vagosympathetic nerve bundles at the level of the carotid sinus bifurcation. Afferent input from carotid sinus baroreceptors was eliminated by bilateral ligation of sutures around the carotid sinus nerve, the internal carotid artery, and the occipital artery complex. The ligation effectively crushed the carotid sinus nerve and eliminated afferent input from carotid baroreceptors (21, 37). The efficacy of denervation was confirmed by the absence of a pressor response to brief bilateral occlusion of the common carotid arteries (MAP = 22 ± 3 and 3 ± 1 mmHg before and after denervation, respectively).

Histological examination. The injection sites were examined after each experiment. A nanoinjector was used to inject 0.1 µl of 2% sky blue dye in PBS into the regions. The brain stem was then removed, fixed in a solution of 10% phosphate-buffered formalin, and stored at 4°C. After the tissue was adequately fixed, the brain stem was blocked and subsequently sliced into 50-µm sections on a cryostat (model 2800 Frigocut-E, Cambridge). The sections were placed on coated slides. Placement of the pipette was verified by using Berman's atlas (2) as a reference. If histological examination showed that micropipettes were located in the lateral PAG, the data obtained from these cats were included in this study.

Data analysis. Control values were determined by analyzing ≥30 s of the data immediately before a given muscle contraction. The peak response of each variable was determined by the peak change from the control value. Experimental data (MAP, HR, and tension) were analyzed by one-way ANOVA with repeated measures. Tukey's post hoc test was utilized as appropriate. Values are means ± SE. For all analyses, differences were considered significant if P < 0.05. All statistical analyses were performed using SigmaStat for Windows version 2.03 (SPSS).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Effect of GABA on the exercise pressor reflex. The effect of GABA in the PAG on the pressor response to 1 min of muscle contraction was examined in six cats (Fig. 1). The basal MAP and HR were not altered significantly before and after bilateral microinjection of 0.1 µl of the GABA receptor agonist muscimol (0.01, 0.1, and 1 mM) into the PAG. After bilateral injection of 1 mM muscimol, the pressor response to contraction was significantly attenuated (peak tension = 5.8 ± 0.8 kg and MAP = 102 ± 12 to 126 ± 14 mmHg) compared with control (peak tension = 5.9 ± 0.6 kg and MAP = 99 ± 10 to 145 ± 12 mmHg). The HR response was also attenuated by injection of muscimol ({Delta}HR = 12 ± 2 vs. 22 ± 4 beats/min in control, P < 0.05). This result suggests that activation of GABA receptors within the PAG plays a role in attenuating the exercise pressor reflex.



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  		Fig. 1. Left: representative traces from 1 cat showing that bilateral microinjection of the GABA receptor agonist muscimol into the lateral periaqueductal gray (PAG) attenuates the pressor response evoked by 1 min of static muscle contraction. Right: dose-response relation showing that 1 mM muscimol significantly attenuates the muscle contraction-mediated pressor response (n = 6). Static muscle contraction was induced by electrical stimulation of L7 and S1 ventral roots. MAP, mean arterial pressure; ABP, arterial blood pressure. *P < 0.05 vs. control and recovery.

 
In addition, the effect of 0.01 and 0.1 mM bicuculline on the pressor response to muscle contraction was also examined. Bicuculline at 0.01 mM had no significant effect on the pressor response to muscle contraction (peak tension = 5.6 ± 0.8 kg and MAP = 96 ± 9 to 146 ± 16 mmHg; Fig. 2B). However, the pressor response to contraction was significantly enhanced by 0.1 mM bicuculline (peak tension = 5.9 ± 0.9 kg and MAP = 105 ± 8 to 170 ± 15 mmHg; Fig. 2B). The HR responses were 18 ± 4 and 26 ± 6 beats/min in control and after injection of 0.1 mM bicuculline, respectively. These results suggest that GABA release within the PAG produces tonic inhibition of the exercise pressor reflex. Furthermore, the effect of 1 mM muscimol was examined with the prior injection of 0.01 mM bicuculline in four cats (Fig. 2A). The prior injection of bicuculline significantly blunted the attenuating effect evoked by muscimol on the exercise pressor reflex (peak tension = 5.6 ± 0.6 kg and MAP = 96 ± 8 to 142 ± 14 mmHg). Bicuculline at 0.01 mM was chosen to determine its antagonizing effect on GABA activation, because the pressor response to contraction was not affected by this dose of bicuculline (Fig. 2B).



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  		Fig. 2. A: effects of prior microinjection of the GABAA receptor antagonist bicuculline (Bicu, 0.01 mM) into the lateral PAG on muscimol (Mus) attenuation of muscle contraction-mediated pressor response (n = 4). B: effects of bilateral microinjection of 0.01 and 0.1 mM bicuculline into the lateral PAG on the pressor response evoked by 1 min of static muscle contraction. *P < 0.05 vs. control and Mus + Bicu (A) and vs. control and 0.01 mM bicuculline (B).

 
Finally, the reflex pressor response was enhanced after denervation of the arterial baroreflex. The MAP and HR responses to muscle contraction (peak tension = 5.8 ± 0.6 kg) were 98 ± 10 to 158 ± 16 mmHg and 165 ± 25 to 192 ± 20 beats/min, respectively (P < 0.05 vs. control before denervation). However, the denervation significantly attenuated the effect of GABAA receptor blockade on the reflex response (Fig. 3A). The MAP and HR responses to muscle contraction (peak tension = 5.7 ± 0.6 kg) were 68 ± 10 mmHg and 28 ± 6 beats/min after bicuculline injection, respectively. These data suggest that arterial baroreflex activation is necessary for GABA in the PAG to modulate the exercise pressor reflex.



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  		Fig. 3. A: effects of GABAA receptor blockade on the muscle contraction-evoked MAP response after bilateral microinjection of 0.1 mM bicuculline into the lateral PAG in baroreceptor-intact and baroreceptor-denervated animals. B: nitric oxide (NO) attenuation on the muscle contraction-evoked MAP response after bilateral injection of L-arginine into the lateral PAG in intact and denervated animals. *P < 0.05 vs. intact.

 
Effect of NO on the exercise pressor reflex. L-Arginine (5 mM) was injected bilaterally into the PAG to test its effect on the pressor response to muscle contraction in eight cats (Fig. 4). L-Arginine significantly attenuated the exercise pressor reflex compared with control. The MAP response to contraction was 103 ± 10 to 148 ± 15 mmHg in control (peak tension = 5.4 ± 0.5 kg) and 105 ± 12 to 131 ± 14 mmHg with L-arginine injection (peak tension = 5.5 ± 0.6 kg). The HR response was also attenuated by injection of L-arginine ({Delta}HR = 10 ± 2 vs. 18 ± 4 beats/min in control, P < 0.05). The NO attenuation was blunted by the prior injection of L-NAME (Fig. 4). Furthermore, the same concentration of D-arginine injected into the PAG did not attenuate the pressor response to muscle contraction (46 ± 12 and 48 ± 14 mmHg in control and after D-arginine, respectively, P > 0.05, n = 3). These findings suggest that NO plays a role in attenuating the exercise pressor reflex in the PAG. In our preliminary study, an inhibitor of neuronal NO synthase, 7-nitroindazole (10 µM), was also injected into the PAG to block effects of L-arginine on the exercise pressor response (n = 3). The antagonizing effect of 7-nitroindazole was the same as that of L-NAME. Attenuation was 45.3% by 7-nitroindazole and 42.2% by L-NAME (P > 0.05). Thus the data of L-NAME (but not 7-nitroindazole) are reported in this study. In addition, the effect of NO attenuation was antagonized by bicuculline before L-arginine (Fig. 4), indicating that NO within the PAG modulates the exercise pressor reflex via GABA mechanisms.



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  		Fig. 4. Bilateral microinjection of the NO physiological precursor L-arginine (L-Arg) into the lateral PAG attenuates the pressor response to 1 min of static muscle contraction. Prior injections of the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) and the GABAA receptor antagonist bicuculline reduce L-arginine attenuation of the muscle contraction mediated-pressor response. A: representative traces from 1 cat. B: average data from 8 cats. *P < 0.05 vs. control, L-Arg + L-NAME, and L-Arg + Bicu.

 
Finally, denervation significantly reduced the effect of NO attenuation on the exercise pressor reflex (Fig. 3B). The MAP and HR responses by muscle contraction (peak tension = 5.4 ± 0.6 kg) were 46 ± 8 mmHg and 18 ± 6 beats/min, respectively, after L-arginine injection.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It has been suggested that the PAG is a neural integrating site for the interaction between the exercise pressor reflex and the arterial baroreflex (21, 22). The purpose of this study was to examine the role of GABA and NO in the PAG in modulating the autonomic responses observed when muscle and baroreceptor reflex inputs are activated. The results demonstrate that the pressor response to muscle contraction was significantly attenuated by activation of GABA receptors in the PAG (Fig. 1). This attenuation was selectively antagonized by 0.01 mM bicuculline (Fig. 2A). It has been reported that bicuculline is a GABAA receptor antagonist (4). Furthermore, the reflex response was significantly enhanced after GABAA receptor blockade (0.1 mM bicuculline; Fig. 2B). The effect of GABAA receptor blockade was significantly reduced after denervation (Fig. 3A). This suggests that engagement of the arterial baroreflex is necessary for GABA in the PAG to modulate the pressor response observed with muscle contraction. In addition, the results from this study further show that the pressor response to contraction was significantly attenuated by microinjection of L-arginine into the PAG (Fig. 4). Importantly, the effect of NO attenuation was antagonized by blockade of the PAG GABAA receptor (Fig. 4). The present data suggest that PAG NO modulates the exercise pressor reflex via GABA mechanisms. It has been reported that NO is involved in PAG cardiovascular regulation (7, 12), and this effect is mediated in part by increasing presynaptic vesicular release of GABA (12). Arterial blood pressure rises with muscle contraction (5, 26, 32), which activates the arterial baroreflex. NO attenuation was blunted after denervation of the arterial baroreflex (Fig. 3B). These data further suggest that PAG GABA-mediated NO attenuation requires engagement of the arterial baroreflex (Fig. 5). Removal of the arterial baroreflex may blunt GABA release in the PAG.



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  		Fig. 5. A major hypothesis that an increase in PAG GABA by arterial baroreceptor reflex (ABR) during muscle contraction attenuates the exercise pressor reflex (EPR) and NO increases GABA to attenuate the EPR. – and +, Inhibition and excitation, respectively; GLU, glutamate.

 
The arterial baroreflex is altered during exercise in humans and animals (9, 39, 40, 46). This observation has been explained as follows: the arterial blood pressure is inoperative or greatly suppressed, or the baroreflex stimulus-response curve is shifted or reset by exercise to a higher set-point pressure (27, 39). The level of baroreceptor input from the isolated carotid sinus region is critical in determining the magnitude of the cardiovascular adjustments during treadmill exercise in dogs (30, 46). When carotid sinus pressure is maintained at a constant level and the arterial baroreflex is unable to respond to changes in blood pressure during exercise, the increases in HR and cardiac output are potentiated compared with the situation when carotid sinus pressure is allowed to follow the rise in exercise blood pressure. Similarly, the response of renal sympathetic nerve activity during exercise is potentiated when the rise in systemic pressure is prevented by infusion of sodium nitroprusside (9). Furthermore, the reflex bradycardia evoked by an increase in carotid sinus pressure is attenuated when accompanied by muscle contraction (28, 29). From this study, it was concluded that the sensitivity of the carotid baroreflex is inhibited by activation of muscle afferents. Moreover, the nature of the interaction between the arterial baroreflex and the exercise pressor reflex is dependent on the level of baroreceptor input (38). A low level of baroreceptor input facilitates the excitatory sympathetic responses evoked by muscle contraction, whereas a high level of input attenuates these responses (38). However, neural mechanisms involved in the processing of inputs from two reflexes remain unclear.

The studies have shown that skeletal muscle and baroreceptor afferent inputs activate neuronal cells in the PAG during muscle contraction (22). Furthermore, anatomic evidence has shown that afferent fiber projections from the first synaptic site receiving skeletal muscle receptor afferents terminate in the PAG neurons (6, 18) that are activated by arterial baroreflex inputs (22, 33). In a previous study (21), muscle contraction was performed in baroreceptor-intact and baroreceptor-denervated cats. Thus muscle and baroreceptor afferent inputs were activated simultaneously in the intact animals, and muscle afferents were activated exclusively in baroreceptor-denervated animals. Additionally, intravenous injections of phenylephrine were used to stimulate the arterial baroreflex in intact cats. The results from this study showed that muscle and baroreceptor afferent inputs separately increased glutamate concentration within the PAG, and the summation of glutamate increases evoked by individual reflex inputs was greater than the change in glutamate noted when both reflexes were activated simultaneously. These findings demonstrate convergence of afferent inputs from muscle receptors and arterial baroreceptors in the PAG (21). Therefore, these studies support the hypothesis that the PAG is a neural integrating site in the central interaction between two reflexes. The results from the latter study (21) also suggest the existence of a mechanism in the PAG by which glutamate increase is inhibited during simultaneous activation of the exercise pressor reflex and arterial baroreflex. It is reasoned that the transmission of skeletal muscle input is inhibited in the PAG by the arterial baroreflex, and this may contribute to the central interaction between the two reflexes. Speculatively, GABA released by the arterial baroreflex during the exercise pressor reflex may inhibit PAG glutamate interneurons or glutamate presynaptic release activated by contracting muscle to reduce the glutamate level in the PAG (Fig. 5). Our present study has demonstrated that activation of GABA receptors in the PAG attenuates the exercise pressor reflex and blockade of GABAA receptor potentiates this reflex, suggesting that PAG GABA release during muscle contraction has an inhibitory effect on the pressor response. These data also support the hypothesis that engagement of the arterial baroreflex is necessary for GABA attenuation, because the effect of GABAA receptor blockade was significantly reduced after denervation.

NO has been reported to be involved in cardiovascular regulation in the PAG (7, 12). The effect of NO within the PAG on the cardiovascular responses is mediated in part by increased presynaptic vesicular release of GABA (12). Anatomic studies have shown that the muscle contraction-activated neurons are in close proximity to neuronal processes containing NO synthase in the PAG (20). The present data demonstrate that L-arginine (a precursor of NO synthesis) microinjected into the PAG attenuates the exercise pressor reflex, and this attenuation was blunted by blockade of the GABAA receptor. This supports the hypothesis that NO attenuates the exercise pressor reflex by increasing GABA release in the PAG. This effect is significantly attenuated by denervation of the arterial baroreflex. These results support the hypothesis that an increase in PAG GABA by the arterial baroreflex during activation of muscle afferents attenuates the exercise pressor reflex and NO attenuates the exercise pressor reflex by involvement of the arterial baroreflex-engaged GABA mechanisms (Fig. 5).

Finally, two potential limitations of the study should be acknowledged. First, competitive binding studies in guinea pig brain tissue have shown an affinity for L-NAME with muscarinic receptors (3), suggesting that L-NAME antagonizes muscarinic receptors. The consequences of L-NAME's functional dichotomy on reflex-mediated cardiovascular regulation within the brain stem remain unknown. However, it has been shown that the blood pressure-raising effects of intracerebroventricular administration of L-NAME are unaffected by coadministration of the muscarinic receptor antagonist methylatropine (35). In the present study, L-arginine injected into the PAG attenuated the exercise pressor reflex, and this effect was reversed by L-NAME. Thus the results of this and previous studies suggest that L-NAME primarily serves to antagonize NO within the PAG. Second, an in vitro study has shown that L-NAME inhibits the reduction of ferric cytochrome c by ferrous iron (36). L-NAME's ability to inhibit electron transfer suggests that it is capable of altering the function of numerous enzymes; therefore, its hemodynamic effects may not be due exclusively to the inhibition of NO synthase. However, similar effects on iron-dependent systems have been reported for the inactive isomer D-NAME. Generally, this has not been the case when physiological in vivo systems have been tested. For instance, L-NAME, but not D-NAME, administration within the brain stem has yielded systemic hemodynamic effects (8) and has altered the function of the baroreflex (13) and the somatosympathetic C-fiber reflex (23) (a known component of the exercise pressor reflex). Therefore, L-NAME's effects on iron-containing systems may have minimal impact on the cardiovascular responses reported in this study.

In conclusion, results of this study show that GABA and NO within the PAG modulate the exercise pressor reflex and that the arterial baroreflex-mediated GABA increase plays a role in the ability of NO to attenuate the exercise pressor reflex. The results suggest that the PAG plays an important role in modulating the cardiovascular responses to activation of muscle afferents, and GABA is a key neurotransmitter in these processes.


   GRANTS
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 ABSTRACT
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 RESULTS
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 GRANTS
REFERENCES
 
The author thanks Dr. Jere H. Mitchell (The University of Texas Southwestern Medical Center at Dallas) for encouragement and generous support (by National Heart, Lung, and Blood Institute Grant HL-06296) and Dr. Lawrence I. Sinoway (Pennsylvania State College of Medicine) for scientific input and generous support (by National Heart, Lung, and Blood Institute Grant R01 HL-60800). This study was partly supported by American Heart Association Texas Affiliate Grant 9960088Y and American Heart Association Grant 0265375U (to J. Li).


   ACKNOWLEDGMENTS
 
The author thanks Margaret Robledo and Julius Lamar, Jr. (University of Texas Southwestern Medical Center) and Val Kehoe (Pennsylvania State College of Medicine) for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Li, Div. of Cardiology H047, Dept. of Medicine, Penn State Univ., The Milton S. Hershey Medical Center, 500 Univ. Dr., Hershey, PA 17033 (E-mail: jzl10{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
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 DISCUSSION
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