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Am J Physiol Heart Circ Physiol 288: H2068-H2076, 2005. First published December 16, 2004; doi:10.1152/ajpheart.00919.2003
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Independent modification of baroreceptor and exercise pressor reflex function by nitric oxide in nucleus tractus solitarius

Scott A. Smith,1 Jere H. Mitchell,2,3 and Jianhua Li4

Departments of 1Health Care Sciences, 2Internal Medicine, and 3Physiology, Harry S. Moss Heart Center, University of Texas Southwestern Medical Center, Dallas, Texas; and 4Division of Cardiology, Department of Medicine, Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Submitted 26 September 2003 ; accepted in final form 13 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It has been suggested that nitric oxide (NO) is a key modulator of both baroreceptor and exercise pressor reflex afferent signals processed within the nucleus tractus solitarius (NTS). However, studies investigating the independent effects of NO within the NTS on the function of each reflex have produced inconsistent results. To address these concerns, the effects of microdialyzing 10 mM L-arginine, an NO precursor, and 20 mM NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, into the NTS on baroreceptor and exercise pressor reflex function were examined in 17 anesthetized cats. Arterial baroreflex regulation of heart rate was quantified using vasoactive drugs to induce acute changes in mean arterial pressure (MAP). To activate the exercise pressor reflex, static hindlimb contractions were induced by electrical stimulation of spinal ventral roots. To isolate the exercise pressor reflex, contractions were repeated after barodenervation. The gain coefficient of the arterial cardiac baroreflex was significantly different from control (–0.24 ± 0.04 beats·min–1·mmHg–1) after the dialysis of L-arginine (–0.18 ± 0.02 beats·min–1·mmHg–1) and L-NAME (–0.29 ± 0.02 beats·min–1·mmHg–1). In barodenervated animals, the peak MAP response to activation of the exercise pressor reflex (change in MAP from baseline, 39 ± 7 mmHg) was significantly attenuated by the dialysis of L-arginine (change in MAP from baseline, 29 ± 6 mmHg). The results demonstrate that NO within the NTS can independently modulate both the arterial cardiac baroreflex and the exercise pressor reflex. Collectively, these findings provide a neuroanatomical and chemical basis for the regulation of baroreflex and exercise pressor reflex function within the central nervous system.

autonomic nervous system; arterial baroreflex

EXERCISE PRODUCES INTENSITY-dependent increases in heart rate (HR) and arterial blood pressure (ABP) via coordinated changes in autonomic outflow. The adjustments in sympathetic and parasympathetic neural control during physical activity are mediated in part by the arterial baroreflex (19, 32), the exercise pressor reflex (a peripheral neural drive that originates in skeletal muscle; Refs. 16, 21), and central command (a central neural drive that originates in higher brain centers associated with the volitional component of exercise; Refs. 6, 11). Although much has been discerned regarding the physiological functions of each of these regulatory inputs during health and disease, relatively little is known about the central nuclei and neurochemicals involved in processing these inputs during exercise.

Recent evidence suggests that the functions of at least two of these control mechanisms, namely, the baroreflex and the exercise pressor reflex, may be modulated by the actions of nitric oxide (NO) within the nucleus tractus solitarius (NTS) of the medulla oblongata (7, 13, 24). However, these studies are not without controversy. For example, experimental augmentation of NO bioavailability within the NTS has in some cases been shown to reduce baroreflex sensitivity (24), whereas in other studies it has had no effect on reflex function (26, 40). With respect to the exercise pressor reflex, NO has been reported to attenuate the increase in mean arterial pressure (MAP) elicited by activation of skeletal muscle afferents during static contractions (13). However, in the latter set of studies, conclusions were derived from experiments in barointact animals. If NO affects baroreflex function as has been suggested (7, 24), the results of such experiments are difficult to interpret. In addition to these concerns, to date, studies have been designed to examine the modulatory effect of NO within the NTS on either the baroreflex or the exercise pressor reflex but not on both within the same preparation. Furthermore, most of these investigations have been conducted in different species, which makes direct comparisons difficult and inferences regarding the interactive relationship between the two reflexes impossible.

To address these concerns, both the baroreceptor and the exercise pressor reflexes were investigated in cats before and after microdialysis of an NO precursor (L-arginine) and a nonselective NO synthase (NOS) inhibitor [NG-nitro-L-arginine methyl ester (L-NAME)] into the NTS. The primary purpose of the investigation was to test the hypothesis that within the same species and preparation, NO within the NTS independently modulates 1) baroreflex function, and 2) exercise pressor reflex function. Collectively, the goal of these experiments was to establish a neuroanatomical and chemical basis for the regulation of baroreflex and exercise pressor reflex function within the central nervous system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General surgical preparation. Experiments were performed on 17 mongrel cats (body wt, 3–5 kg) of either sex. The procedures outlined were approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Anesthesia was initially induced with isoflurane gas (2–5% in 100% oxygen). A jugular vein and the left femoral artery were cannulated for drug administration and blood pressure measurement, respectively. Anesthesia was subsequently maintained by infusion of {alpha}-chloralose (80 mg/kg iv) and urethane (200 mg/kg iv). Animals were intubated and artificially ventilated with a mechanical respirator (model 661, Harvard Apparatus). Supplemental doses of {alpha}-chloralose (15 mg/kg) and urethane (40 mg/kg) were administered as deemed necessary by a withdrawal reflex to pinching of the hindpaw, presence of a corneal reflex, and/or spontaneous increases in HR or ABP throughout the experiment. Arterial blood gas levels were periodically monitored using an automated blood gas analyzer (model ABL5, Radiometer) and were maintained within normal ranges (arterial PO2, >80 mmHg; arterial PCO2, 35–45 mmHg; pH, 7.3–7.4). Body temperature, as assessed by rectal thermometer, was kept within 37–38°C by a water-perfused heating pad and an external heat lamp. At the conclusion of each experiment, animals were humanely euthanized by administration of pentobarbital sodium (120 mg/kg iv).

In a subset of animals (n = 6), additional surgery was performed to allow 1) acute sinoaortic denervation and vagotomy, and 2) electrically induced static muscle contraction. With the use of a ventral approach, the common carotid artery and carotid sinus bifurcation were surgically exposed on each side of the neck. Silk sutures were placed around the carotid sinus nerve, the internal carotid artery, and the occipital artery distal to the bifurcation. Sutures were also placed around the vagosympathetic nerves bilaterally. Additionally, a dorsal laminectomy was performed exposing the seventh lumbar (L7) and first sacral (S1) portions of the spinal cord. The L7 and S1 ventral roots were cut, and the peripheral ends were placed on bipolar stimulating electrodes and immersed in warmed (37°C) mineral oil. The calcaneal bone of the right hindlimb was sectioned, and the Achilles tendon was connected to a force transducer.

Microdialysis procedures. All animals were held in a stereotaxic head unit and spinal frame (Kopf Instruments). A limited occipital craniotomy was performed to expose the dorsal surface of the brain stem. A microdialysis probe (0.5 mm od, 1 mm membrane length; model CMA 10, Bioanalytical Systems) was stereotaxically positioned (1) within the NTS (coordinates: 1.5 mm lateral to midline, 1.0 mm rostral to obex, and 1.5 mm below dorsal medullary surface). Placement coordinates were carefully chosen to correspond to areas within the NTS that are known to receive afferent projections from both the baroreceptor and exercise pressor reflexes (12, 14). A representative example of the location of the microdialysis probe is shown in Fig. 1, A and B. Probes were placed unilaterally to reduce surgical trauma within the medulla in an attempt to preserve the structural and functional integrity of the brain stem throughout the duration of the protocols. Probes were continuously perfused at a rate of 5 µl/min with artificial extracellular fluid (ECF) buffered to a pH of 7.4. ECF, made fresh for each experiment, contained 0.2% bovine serum albumin, 0.1% bacitracin, and the following ions (in mM): 6.2 K+, 134 Cl–, 2.4 Ca2+, 150 Na+, 1.3 P–, 13 HCO3–, and 1.3 Mg2+. After the probe was inserted, the preparation was allowed to stabilize for a minimum of 1 h.



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  		Fig. 1. Microdialysis probe placement in one representative animal. A: dorsal medulla. Probe was placed 1.5 mm lateral to the midline, 1.0 mm rostral to the obex, and 1.5 mm below the medullary surface (position indicated by black circle). B: spread of Evans blue dye (shaded area) within the nucleus tractus solitarius (NTS) of the medulla. C: photomicrograph of the medulla from one animal. Circumscribed area marks the region stained by perfusing the dialysis probe with 2% Evans blue dye. Minimal structural damage was caused by placement of the dialysis probe (arrow); 4V, fourth ventricle.

 
Subsequently, animals were divided into two groups, and distinct experimental procedures were performed on each group. Animals not undergoing surgical preparation for barodenervation and static muscle contraction were assigned to protocol 1. Those undergoing this surgery were assigned to protocol 2.

Protocol 1: baroreflex testing. In eight cats, three doses each of the vasoactive drugs phenylephrine (PE; range, 18–65 µg/ml) and sodium nitroprusside (SNP; range, 4–15 µg/ml) were administered randomly to acutely manipulate ABP during the individual dialysis of 1) ECF (control), 2) 10 mM L-arginine, and 3) 20 mM L-NAME into the NTS. ECF, L-arginine, and L-NAME were dialyzed for a minimum of 40 min before drug administration and dialysis continued until the testing cycle was completed. Previously, these dialysis agents have been shown (13) to be maximally effective at these doses within this time frame. Baseline hemodynamics were determined by analyzing 30 s of data immediately before a given drug injection. The maximal response for each variable, which was defined as the greatest change from baseline elicited by the vasoactive substance, was recorded and used for statistical analysis. A minimum of 15 min elapsed between injections to allow drug clearance and cardiovascular stabilization. Data collected from execution of these procedures characterized the closed-loop stimulus-response relationship for the arterial baroreflex control of HR. A summary of this protocol is provided in Table 1.


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  		Table 1. Summary of experimental protocols

 
Protocol 2: exercise pressor reflex testing. The circulatory response to electrically induced static muscle contraction was obtained during the individual administration of ECF (control), 10 mM L-arginine, and 20 mM L-NAME in six cats. In these experiments, the microdialysis probe was placed ipsilateral to the contracting muscle. Furthermore, the concentrations of dialysate used were carefully chosen, as these have been previously shown (13) to alter exercise pressor reflex function. ECF, L-arginine, and L-NAME were dialyzed a minimum of 40 min before any contractile event, and dialysis continued until the testing cycle was completed. Baseline hemodynamics were determined using 30 s of data before contraction. The peak cardiovascular response was defined as the largest change from baseline elicited during a 1-min contractile period. During dialysis of each substance, two reproducible responses were obtained with a minimum of 30 min between contractile events. Static contraction of the triceps surae muscles was induced by electrical stimulation (parameters: 3x motor threshold, 0.1 ms duration, and 40 Hz) of the L7 and S1 ventral roots (S88 stimulator, Grass Instruments). Hindlimb muscle was preloaded with 0.4 kg of tension before any manipulation. Previously, this technique has been shown (22) to engage the exercise pressor reflex independent of input from central command. A summary of this protocol is provided in Table 1.

Trials were first executed with the baroreflex intact. Subsequently, contractile protocols during ECF, L-arginine, and L-NAME dialysis were repeated at a minimum of 60 min postbarodenervation. The vagosympathetic trunks were ligated and cut bilaterally to eliminate input from aortic and cardiopulmonary baroreceptor afferents. Owing to physical and mechanical limitations presented by the stereotaxic head unit, it was not possible to section the carotid sinus nerve. To reduce input from the carotid baroreflex, sutures placed around the carotid sinus nerve, internal carotid artery, and occipital artery were ligated bilaterally. The effectiveness of this procedure was confirmed by ≥75% reduction in the pressor response to vascular occlusion of the common carotid arteries (a stimulus that deactivated carotid sinus baroreceptors). Data collected from these experiments characterized exercise pressor reflex-mediated control of HR and ABP in the presence and absence of baroreceptor input.

In a corollary experiment (n = 3 cats), exercise pressor reflex function was examined before and after the microdialysis of 7-nitroindazole (7-NI), a selective inhibitor of the neuronal isoform of NOS. After we determined the response to activation of the exercise pressor reflex during the administration of ECF (i.e., control), graded concentrations of 7-NI (10 µM to 1 mM) were dialyzed into the NTS. Each concentration of 7-NI was administered for 40 min. At the end of the 40-min dialysis period for each concentration of 7-NI, the exercise pressor reflex was activated by static muscle contraction as previously described. ECF was readministered for 40 min after 7-NI was discontinued. Subsequently, L-arginine (10 mM) was dialyzed into the NTS for a period of 40 min, and the contraction protocol was repeated. Finally, 7-NI (100 µM) was co-administered with L-arginine (10 mM) into the NTS, and the response to static muscle contraction was retested.

Validation of probe placement. At the conclusion of each experiment, Evans blue dye was microdialyzed into the NTS for 40 min. The brain stem was then excised and fixed in 10% phosphate-buffered formalin and stored at 4°C. Subsequently, medullary tissue was blocked, and 40-µm sections were cut serially using a cryostat (model 2800, Cambridge Instruments). Sections were placed on coated slides and examined for the neuroanatomical location of the probe. The perfusion area of the probe was verified by the distribution of the dye. A representative example of the dye distribution marking the perfusion area of the probe is shown in Fig. 1C.

Data acquisition and statistical analyses. ABP was measured from the femoral artery by a pressure transducer (model P23 ID, Statham). MAP was obtained by integrating the arterial signal with a time constant of 4 s. HR was derived from the ABP pulse using a biotachometer (Gould Instruments). Contraction-induced tension development was quantified using a force transducer (model FT10, Grass Instruments). All data were subjected to analog-to-digital conversion (micro 1401, Cambridge Electronic Design) using commercially available software (Spike 2 version 3, CED) and recorded on a personal computer (550-MHz Pentium III, Dell Computer). Statistics were performed using linear regression analysis or repeated-measures ANOVA with a Student-Newman-Keuls post hoc test employed as appropriate. All statistical tests were performed using SigmaStat 2.03 for Windows (SPSS). Results are presented as means ± SE. The level of significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Modulation of baroreflex function. Under control conditions (i.e., microdialysis of ECF; n = 8 cats), baseline HR and MAP were 163 ± 10 beats/min and 110 ± 6 mmHg, respectively. These basal values were not altered by the dialysis of L-arginine or L-NAME. Furthermore, there were no significant differences in baseline hemodynamic parameters before drug injection at any concentration administered. PE- and SNP-induced alterations in MAP (approximate range, ±60 mmHg) elicited significant changes in HR during all trials (Fig. 2A). The baroreflex gain coefficient (i.e., slope), determined from linear regression analyses, was significantly (P = 0.02) reduced during microdialysis of the NO precursor L-arginine. Conversely, diminutions in NO production by dialysis of L-NAME significantly (P < 0.05) potentiated the sensitivity of the baroreflex (Fig. 2B).



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  		Fig. 2. Arterial baroreflex control of heart rate (HR) in response to acute alterations in mean arterial pressure ({Delta}MAP). A: changes in cardiac chronotropy ({Delta}HR), evoked by intravenous administration of pressor (phenylephrine) and depressor (sodium nitroprusside) agents, during microdialysis of either extracellular fluid (ECF; control), L-arginine, or NG-nitro-L-arginine methyl ester (L-NAME) in the nucleus tractus solitarius; bpm, beats/min. B: baroreflex gain as estimated from linear regression analyses. For regression analysis, all data points within a trial were used for statistical characterization of each animal. Means were obtained, as presented in A, for presentation only; P < 0.001 and r > 0.83 for all regressions. *P < 0.05 vs. control; {dagger}P < 0.05 vs. L-arginine.

 
Modulation of exercise pressor reflex function by L-arginine and L-NAME. The peak cardiovascular responses to electrically induced static contraction in barointact and barodenervated cats (n = 6 animals) are presented in Table 2. Baseline hemodynamics were similar under all experimental paradigms and remained unchanged after sinoaortic deafferentation. Activation of skeletal muscle afferent fibers by muscle contraction elicited increases in MAP, HR, and tension that were significantly (P < 0.05) greater than basal values under all dialysis conditions. Importantly, the amounts of tension developed during contraction were similar between trials and conditions, which precluded muscle fatigue as a factor in determining circulatory responsiveness to a given manipulation.


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  		Table 2. Cardiovascular responses to contraction in barointact and barodenervated cats

 
Compared with the elevations in HR evoked by control contractions (i.e., during microdialysis of ECF), microdialysis of L-arginine and L-NAME into the medullary NTS produced negligible nonsignificant changes in the HR increase mediated by activation of the exercise pressor reflex (Fig. 3A). A similar chronotropic response pattern was elicited by static muscle contraction after barodenervation during dialysis of these substances (Fig. 3B). In contrast, the pressor response to exercise was significantly (P < 0.05) attenuated during the dialysis of L-arginine compared with control ({Delta}MAP, 25 ± 10 vs. 43 ± 7 mmHg, respectively) in animals with preserved baroreflex function (Fig. 3C). After sinoaortic deafferentation, the significant (P < 0.05) reduction in pressor reflex circulatory control remained (Fig. 3D). However, in the latter condition, the magnitude of this reduction was significantly (P < 0.05) smaller than in the barointact state (Fig. 4A). For comparison, with normal baroreflex function, the pressor response elicited by static muscle contraction during perfusion of L-arginine was 55% of the control response. After elimination of baroreceptor input, the response was 74% of the control response. Conversely, dialyzing the NOS inhibitor L-NAME affected exercise pressor reflex function minimally by producing slight and insignificant elevations in the contraction-induced MAP response after barodenervation only (see Fig. 3, C and D and Fig. 4B). The efficacy of the barodenervation procedure was quantified in all experiments in which this strategy was employed. In barointact animals, bilateral occlusion of the common carotid arteries increased MAP by 29 ± 8 mmHg. This response was reduced by ~80% ({Delta}MAP, 6 ± 4 mmHg) after barodenervation.



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  		Fig. 3. Cardiovascular responses to activation of skeletal muscle afferents before and after sinoaortic barodenervation. A and B: HR changes evoked by static muscle contraction in barointact (A) and barodenervated (B) cats during the microdialysis of ECF (control), L-arginine (10 mM), or L-NAME (20 mM) into the NTS. C and D: MAP responses to static muscle contraction before (C) and after (D) barodenervation under control conditions and during brain-stem dialysis of L-arginine or L-NAME. First and second bars for the L-arginine and L-NAME trials represent contractile events at 40 and 70 min, respectively, after the microdialysis procedure was started for each substance. For a given substance, the responses elicited at these time points were not different, which established temporal reproducibility. *P < 0.05 vs. control.

 


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  		Fig. 4. Differential exercise pressor reflex control of MAP in barointact and barodenervated cats. A: effects of dialyzing the nitric oxide (NO) precursor L-arginine in the NTS on the pressor response evoked by static muscle contraction. B: changes in MAP response to static muscle contraction after NTS perfusion of the NO synthase inhibitor L-NAME. Data are expressed as differences from control responses. Time indicates the period between the beginning of dialysis of either L-arginine or L-NAME and the contractile event. Under each condition (i.e., barointact or barodenervated), the responses elicited at these time points for a given dialysate were not different, thereby establishing temporal reproducibility. *P < 0.05 vs. barointact state.

 
Modulation of exercise pressor reflex function by 7-NI. The effects of microdialyzing 7-NI into the NTS on the MAP response to activation of the exercise pressor reflex (n = 3 cats) are presented in Fig. 5. The administration of 10 µM, 100 µM, and 1 mM 7-NI into the NTS did not significantly alter the pressor response to static muscle contraction from values obtained under control (i.e., ECF) conditions (Fig. 5A). Furthermore, the MAP response to contraction during dialysis of the selective neuronal NOS inhibitor 7-NI was not different than that elucidated during dialysis of the nonselective NOS inhibitor L-NAME (Fig. 5B); however, 7-NI did antagonize the blunting effects of L-arginine on the pressor response to static muscle contraction (Fig. 5C). The dialysis of 7-NI within the NTS had no effect on the HR response to muscle contraction during any trial. The amount of tension developed during contraction (~4.0 kg) was not different between trials.



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  		Fig. 5. Effects of the neuronal NOS inhibitor 7-nitroindozale (7-NI) on the pressor response to static muscle contraction. A: MAP changes evoked by static muscle contraction during the microdialysis of ECF (control and recovery) and 7-NI into the NTS. B: pressor responses to activation of the exercise pressor reflex by muscle contraction during the dialysis of ECF, L-NAME, and 7-NI. In B, 7-NI was dialyzed in a different set of animals than those administered L-NAME. C: MAP response to contraction during the individual dialysis of ECF and L-arginine (L-Arg) and during the co-administration of L-arginine and 7-NI. Dialysis of 7-NI within the NTS had no effect on the HR response to muscle contraction during any trial. Amount of tension developed during contraction (~4.0 kg) was not different between trials. *P < 0.05 vs. control and L-Arg + 7-NI.

 
Microdialysis probe placement and diffusion distance. Structural damage to the brain stem was minimized by gently advancing the dialysis probe anteriorly to the NTS using a micromanipulator. Histological examination of tissue sections revealed that the tract produced by the probe extended ~1.5 mm in depth from the dorsal surface of the medulla (see Fig. 1C). Examination also confirmed that the spread of Evans blue dye was restricted within the anatomical borders of the NTS.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The cardiovascular response to exercise is mediated in part by input from receptors that originate in large arterial vessels (carotid sinus and aortic arch baroreflexes) and skeletal muscle (exercise pressor reflex). The site of neural integration and the mechanism of chemical processing of these afferent inputs remain areas of controversy and intense investigation. To address this controversy, we assessed the role of NO within the NTS in the regulation of both baroreflex and exercise pressor reflex functions. The most important findings from this investigation were derived under conditions in which each reflex was isolated experimentally and include 1) increasing NO production in the NTS reduced the sensitivity of the arterial cardiac baroreflex, whereas blockade of NO induced the opposite effect; and 2) increasing NO production in the NTS attenuated exercise pressor reflex-mediated increases in arterial pressure during static muscle contraction. These findings establish for the first time in the same species and preparation that NO within the NTS maintains the potential to modify both baroreceptor and skeletal muscle afferent inputs independently. Collectively, the data implicate the NTS as a significant central processing center and NO as an important modulator of both the baroreceptor and exercise pressor reflexes.

NO, NTS, and arterial baroreflex control of HR. The modulatory capacity of NO within the NTS on the baroreflex control of HR has not been clearly established, and studies often report conflicting results. For example, it has been shown in conscious rats (26) that both acute blockade of NOS and/or increased production of NO in the NTS have no effect on the gain of the arterial cardiac baroreflex. More recently, Talman and Dragon (34) have reported that preferential blockade of the neuronal isoform of NOS significantly reduced the gain of the arterial cardiac baroreflex in chloralose-anesthetized rats. In contrast, it has been shown (38) that chronic inhibition of the endothelial isoform of NOS using a recombinant adenoviral vector within the NTS increases the gain of the cardiac baroreflex in conscious rats. Furthermore, Paton et al. (24) have determined in the decerebrate rat that acute NTS microinjection of an NO donor depresses the gain of the cardiac component of the baroreflex. The discrepancies among studies are not readily apparent and most likely result from differences in the use (chloralose) or nonuse (decerebrate, conscious) of anesthesia as well as the method of manipulating NO activity (nonselective vs. selective NOS inhibition, acute vs. chronic alteration of NOS activity). Whether the former or latter findings apply to larger mammalian species such as felines remains undetermined.

In light of this controversy and lack of information in larger animals, we assessed the role of NO within the NTS on the baroreflex control of HR in cats. It was determined from these experiments that dialysis of L-arginine and L-NAME into the NTS significantly altered the gain of the arterial cardiac baroreflex. These findings suggest that in cats, baroreflex control of HR is altered not only under conditions in which NO bioavailability is increased by an exogenous source but also when its endogenous production is blocked. As stated, similar findings have been reported for conscious (38) and decerebrate rats (24) and, therefore, the modulation of the arterial cardiac baroreflex by the actions of NO within the NTS appears to be preserved across species. It should be noted, however, that the technique implemented in this investigation to activate and unload the arterial baroreceptors could only be used to assess the baroreflex control of HR, because arterial pressure was experimentally manipulated by pharmacological agents (i.e., PE and SNP). Therefore, the baroreflex control of the peripheral vasculature could not be determined using this technique. As such, additional experimentation is warranted to determine the effects of NO within the NTS on the baroreflex control of blood pressure.

NO, NTS, and exercise pressor reflex control of HR and MAP. The present study was designed to allow preferential activation of the exercise pressor reflex independent of input from central command and the arterial baroreflex. This was achieved by inducing static muscle contraction by electrical stimulation of spinal ventral roots L7 and S1 (eliminating central command) in barodenervated animals. Under these conditions, increasing the bioavailability of NO via an exogenous source (i.e., L-arginine) in the NTS attenuated the pressor response to static muscle contraction. This finding agrees with a previous report by Li and Potts (13) in which cats were studied. However, in that study, the role of NO within the NTS in modulating the pressor reflex was investigated in animals in which the baroreflex remained intact. As such, it could not be definitively determined whether the attenuation in the pressor response to exercise was mediated by the ability of NO to modulate the baroreflex, the exercise pressor reflex, or both. The present study clearly exhibits the ability of NO within the NTS to alter exercise pressor reflex control of blood pressure during exercise independent of central command or baroreflex input. However, the impact of this finding is tempered by the inability of L-NAME, which blocks endogenous sources of NO, to likewise significantly alter pressor reflex function. This may have stemmed from a technical limitation of the study that restricted delivery of L-NAME to one side of the brain stem rather than bilaterally. In support of this contention, bilateral infusion of 20 mM L-NAME in the NTS has previously been shown (13) to enhance the pressor response to static muscle contraction with the baroreflex intact.

Processing baroreceptor and exercise pressor reflex signals within NTS. Several investigations have implicated the NTS as the potential site for the independent modification as well as integration of baroreflex and exercise pressor reflex input. Nerve degeneration studies (20), transganglionic transport of horseradish peroxidase (9), anterograde tract tracing with biotinylated dextran amine (28), and c-Fos protein immunohistochemistry (3, 14) have all been used to successfully map baroreceptor and skeletal muscle somatosensory afferents to the NTS. In addition, using electrophysiological techniques, Toney and Mifflin (35–37) have demonstrated that excitatory responses can be elicited from NTS neurons in response to direct activation of skeletal muscle afferents by various modalities (e.g., muscle contraction, muscle stretch, intraarterial injection of capsaicin). Furthermore, these excitatory responses undergo significant time-dependent inhibition after experimental stimulation of baroreceptor afferent neurons (35). In contrast, others have identified distinct populations of NTS neurons that are activated by either baroreceptor or skeletal muscle afferent inputs but not both (30). Collectively, these findings suggest that the NTS may serve as a neural substrate for both the independent modification and integrative processing of baroreceptor and skeletal muscle somatosensory input during exercise. The findings of this investigation support this hypothesis.

Processing baroreceptor and exercise pressor reflex inputs: role of NO. Increased production of NO within the NTS has been shown to attenuate the exercise pressor reflex in barointact animals (13). Accumulating evidence suggests that the baroreflex and the exercise pressor reflex modify one another functionally (5, 8, 17, 18, 23, 27, 29, 31, 33). Therefore, it is logical to suggest that the NO-mediated decrease in pressor reflex function reported previously may be facilitated by input from the arterial baroreflex. Supporting this theory, in the present study, the reduction in the pressor response to static muscle contraction during dialysis of the NO precursor L-arginine was significantly greater in animals with preserved baroreflex function compared with those that had been barodenervated. However, even after barodenervation, dialysis of L-arginine within the NTS significantly blunted the exercise pressor reflex. Thus NO-mediated reductions in the exercise pressor reflex can occur independently of baroreflex input. This may explain why under control conditions (i.e., ECF dialysis), barodenervation had no effect on the pressor response to static muscle contraction. If the baroreflex was supplying an endogenous source of NO to attenuate the pressor reflex, then removal of this input by denervation should have augmented the MAP response to muscle contraction. It should be noted, however, that other studies (39) have shown that barodenervated cats exhibit a larger pressor response to static muscle contraction than barointact animals. These findings support a more active role for the baroreflex in facilitating the inhibitory actions of NO on the exercise pressor reflex than reported in the present investigation. Because the difference between these studies is not readily apparent, additional investigation is warranted before definitive conclusions can be made about the interactive relationship between the baroreceptor and exercise pressor reflexes with respect to NO within the NTS.

Experimental limitations. The finding that the baroreflex appeared to facilitate the inhibitory effect of NO on the exercise pressor reflex is puzzling given the fact that NO directly attenuated the baroreflex itself. One explanation for this paradox lies in the design of the present study. By using pharmacological agents with powerful vasoactive effects to acutely alter systemic pressure, we could only assess arterial cardiac baroreflex function. Speculatively, it is possible that NO does not alter the baroreflex control of blood pressure. Therefore, the ability of the baroreflexes to facilitate inhibition by NO of the exercise pressor reflex may stem from the differential effects of NO on the baroreflex control of HR and MAP. This may also explain why barodenervation only affected the exercise pressor reflex control of MAP and not HR during the dialysis of L-arginine. Alternatively, these paradoxical findings could stem from the microdialysis procedures used in this study. The NTS is known to receive, relay, and process sensory information from numerous viscerosomatic inputs including chemoreceptors, skeletal muscle receptors, arterial baroreceptors, and cardiopulmonary baroreceptors (10, 15). Although Evans blue dye staining confirmed that the perfusion area of the microdialysis probe was constrained to the NTS, we cannot exclude the possibility that regions of the NTS other than those directly innervated by arterial baroreceptor and skeletal muscle receptor afferents were affected. Therefore, the results reported in this investigation may represent the effects of NO within the NTS on multiple reflex pathways.

Limitations in the techniques used to assess arterial baroreflex function are acknowledged. Baroreflex testing was designed to induce dynamic and moderately transient changes in arterial pressure to obviate concerns of rapid reflex adaptation or resetting noted with sustained pressure manipulations (4). HR responses elicited under these conditions are predominately mediated by the parasympathetic nervous system, because the brevity of the procedure does not allow the sympathetic component of the response to completely develop. In addition, SNP induces systemic hypotension by virtue of being a peripheral NO donor. The changes in baroreflex function attributed to the actions of L-arginine within the NTS could have been influenced by the actions of SNP-derived NO acting within the NTS or other brain-stem nuclei. However, this possibility is unlikely, because L-NAME, which was restricted to the NTS, increased baroreflex sensitivity during systemic administration of SNP. If NO bioavailability had been significantly increased throughout the brain stem by the administration of SNP, then such a limited application of L-NAME (a NOS inhibitor) would be expected to have no effect. Furthermore, microdialysis of both L-arginine and L-NAME within the NTS altered baroreflex sensitivity when PE (a non-NO donor) was administered systemically. Nevertheless, interpretation of all data should take these limitations into account.

Other potential limitations to the design of this study are recognized. To begin, a nonselective NOS inhibitor (i.e., L-NAME) was used to probe the regulatory role of endogenous NO on exercise pressor reflex function. Although this strategy maintains the potential to identify NO as a modulator of neuronal activity within the NTS, it does not allow determination of the source of NO (e.g., endothelial, neuronal, or inducible NOS). In an attempt to address this concern, we administered 7-NI (a selective neuronal NOS inhibitor) into the NTS in a subset of animals. Similarly to L-NAME, 7-NI by itself had no effect on the pressor response to static muscle contraction at any concentration tested. However, 7-NI did prevent the reduction in exercise pressor reflex function induced by L-arginine. This suggests that the metabolic conversion of L-arginine to L-citrulline and NO, which depresses exercise pressor reflex function, is most likely mediated by the neuronal isoform of NOS. It should be noted that due to technical and practical difficulties, we were not able to probe the effects of 7-NI on baroreflex function. Therefore, we were unable to determine the source of NO that modifies baroreflex function within the NTS. Furthermore, the effects of L-NAME on baroreflex function must be interpreted with caution, because alkyl esters of arginine such as L-NAME have been shown to antagonize muscarinic receptors (2) as well as inhibit the reduction of ferric cytochrome c (25), both of which could modify cardiovascular hemodynamics independent of alterations in NOS activity.

In summary, by using a cat model of static exercise in combination with brain-stem microdialysis techniques, we have established a neuromodulatory role for NO in the central processing of both the baroreceptor and exercise pressor reflexes independently within the same preparation. These findings implicate a potential neuroanatomical and chemical basis for the regulation of each reflex within the central nervous system.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by American Heart Association, Texas Affiliate Grant 9960088Y (to J. Li) and National Institutes of Health Grant HL-06296 (to J. H. Mitchell).


   ACKNOWLEDGMENTS
 
The authors greatly appreciate the expert technical assistance provided by Margaret Robledo and Julius Lamar, Jr.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Li, Division of Cardiology, H047, Dept. of Medicine, Pennsylvania State Univ. College of Medicine, Milton 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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