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Am J Physiol Heart Circ Physiol 278: H1775-H1782, 2000;
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Vol. 278, Issue 6, H1775-H1782, June 2000

NTS A2a purinoceptor activation elicits hindlimb vasodilation primarily via a beta -adrenergic mechanism

Amy M. Kitchen, Tadeusz J. Scislo, and Donal S. O'Leary

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we have shown that activation of adenosine A2a receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of the selective A2a receptor agonist CGS-21680 elicits potent, dose-dependent decreases in mean arterial pressure and preferential, marked hindlimb vasodilation. Although A2a receptor activation does not change lumbar sympathetic nerve activity, it does markedly enhance the preganglionic adrenal sympathetic nerve activity, which will increase epinephrine release and could subsequently elicit hindlimb vasodilation via activation of beta 2-adrenergic receptors. Therefore we investigated whether this hindlimb vasodilation was due to neural or humoral mechanisms. In chloralose-urethan-anesthetized male Sprague-Dawley rats, we monitored cardiovascular responses to stimulation of NTS adenosine A2a receptors (CGS-21680, 20 pmol/50 nl) in the intact control animals; after pretreatment with propranolol (2 mg/kg iv), a beta -adrenergic antagonist; after bilateral lumbar sympathectomy; after bilateral adrenalectomy; and after combined bilateral lumbar sympathectomy and adrenalectomy. After beta -adrenergic blockade, stimulation of NTS adenosine A2a receptors produced a pressor response and a hindlimb vasoconstriction. Lumbar sympathectomy reduced the vasodilation seen in the intact animals by ~40%, and adrenalectomy reduced it by ~80%. The combined sympathectomy and adrenalectomy virtually abolished the hindlimb vasodilation evoked by NTS A2a receptor activation. We conclude that the preferential, marked hindlimb vasodilation produced by stimulation of NTS adenosine A2a receptors is mediated by both the efferent sympathetic nerves directed to the hindlimb and the adrenal glands via primarily a beta -adrenergic mechanism.

adrenal gland; sympathetic nervous system; epinephrine; adenosine; purines; nucleus tractus solitarii


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE NUCLEUS TRACTUS SOLITARII (NTS) is a major integrative center in the brain stem involved in reflex cardiovascular control and coordination of autonomic function (10). The NTS contains a rich vocabulary of neurotransmitters/neuromodulators involved in the processing of visceral and somatic afferent information (18). Recent studies strongly suggest that adenosine may contribute importantly in the integration of afferent information and subsequent alterations in autonomic activity. Adenosine levels in the NTS and other central structures can increase during periods of physiological stress such as hemorrhage, hypoxia, and ischemia (23, 33, 35). Stimulation of presynaptic adenosine A2a receptors activates adenylate cyclase, which enhances Ca2+ influx and, consequently, enhances release of L-glutamate (9, 20), the primary neurotransmitter released from baroreceptor afferents in the NTS (10, 18). Blockade of adenosine A2a receptors in the NTS also decreases the sensitivity of baroreflex control of heart rate (HR) (21). The importance of adenosine in the NTS for the regulation of HR and mean arterial pressure (MAP) is further supported by observations that spontaneously hypertensive rats exhibit an attenuated depressor response to intra-NTS injections of adenosine compared with normotensive rats (32).

Microinjection of adenosine into the caudal and subpostremal NTS decreases MAP, HR, and efferent sympathetic nerve activity (1, 7, 20, 21, 31, 32). This depressor action of adenosine is mediated by A2a receptors (3). Recent studies from our laboratory (5, 27, 28) have shown that selective activation of adenosine A2a receptors in the subpostremal NTS elicits dose-dependent, differential regional vasodilation and sympathetic neural responses. Although stimulation of NTS A2a adenosine receptors evoked a large preferential hindlimb vasodilation, compared with mesenteric and renal vascular beds (5), adenosine A2a stimulation caused no change in the lumbar sympathetic nerve activity (LSNA) directed to the hindlimb (27). The lack of change in LSNA that we observed previously (27) could be explained by an increase in vasodilator activity combined with a simultaneous decrease in the vasoconstrictor nerve activity. Most recently, we observed that preganglionic adrenal sympathetic nerve activity (pre-ASNA), which is directed to the adrenal medulla, is markedly enhanced by stimulation of A2a purinoceptors in the subpostremal NTS (28), whereas renal sympathetic nerve activity (RSNA) and postganglionic adrenal sympathetic nerve activity (post-ASNA) were decreased. Epinephrine released from the adrenal gland can act directly on the beta 2-adrenergic receptors located preferentially in skeletal muscle (34) to produce vasodilation. There is also evidence (8) that epinephrine released from the adrenal gland can be preabsorbed by sympathetic nerve terminals and rereleased as an active sympathetic vasodilator.

Thus the purpose of the present study was to evaluate the relative contributions of humoral and neural mechanisms to the active hindlimb vasodilation elicited by NTS adenosine A2a receptor stimulation. Because both may operate via a beta -adrenergic mechanism (8, 34), we also evaluated the overall importance of the beta -adrenergic receptors in mediating this marked hindlimb vasodilation. We asked three specific questions. 1) Do the peripheral beta -adrenergic receptors participate in the marked hindlimb vasodilation evoked by NTS A2a purinoceptor activation? 2) Do the efferent lumbar sympathetic nerves directed to the hindlimb contribute to this vasodilation? 3) Do the adrenal glands participate in the hindlimb vasodilation elicited by stimulation of NTS adenosine A2a receptors?


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals endorsed by the American Physiological Society and published by the National Institutes of Health.

Design. The contribution of beta -adrenergic receptors, the sympathetic nervous system, and the adrenal glands to the mechanisms mediating the large preferential hindlimb vasodilation following activation of the subpostremal NTS adenosine A2a receptors was studied in 42 male Sprague-Dawley rats (350-400 g). NTS adenosine A2a receptors were activated via microinjection of CGS-21680, a selective adenosine A2a receptor agonist. Eight animals served as the intact controls, eight were pretreated with propranolol (a beta -adrenergic receptor antagonist), ten received bilateral lumbar sympathectomy from the level of L1-L6, nine received bilateral adrenalectomy, and seven animals received both bilateral lumbar sympathectomy and bilateral adrenalectomy.

Instrumentation and measurements. The rats were anesthetized with a combination of alpha -chloralose (80 mg/kg) and urethan (500 mg/kg) administered intraperitoneally, intubated endotracheally, and allowed to respire spontaneously. Rectal temperature was maintained between 37 and 38°C by a water-heating pad (model TP-500, Gaymar Industries). A catheter (PE-50) was placed in the right carotid artery and connected to a TXX-R Viggo-Spectramed pressure transducer to monitor arterial pressure. Two catheters (PE-50) were placed in the right jugular vein to continuously infuse anesthesia (alpha -chloralose, 8-16 mg · kg-1 · h-1, and urethan, 50-100 mg · kg-1 · h-1, approx 0.5-1 ml/h) and to administer drugs. A midline abdominal incision was made and the right common iliac artery exposed. A pulsed Doppler blood flow transducer (Crystal Biotech) was placed around the artery. In addition, 10 animals underwent bilateral lumbar sympathectomy. The sympathetic trunks and their sympathetic ganglia were isolated and removed from the level of the renal vein down past the aortic bifurcation. In nine animals the adrenal glands were isolated, ligated, and removed. In seven animals both bilateral lumbar sympathectomy and adrenalectomy were performed. The incision was then closed in layers, and the flow probe wires were exteriorized at the point of incision and subsequently attached to a pulsed Doppler flowmeter (Baylor Electronics). The pressure transducer and flowmeter were connected to a Beckman Dynograph (R711). These signals were also transmitted to an analog-to-digital converter (Modular Instruments) interfaced to a laboratory computer. HR was calculated by the computer using either the arterial pressure or flow pulsatile waveform. All variables were recorded continuously using Biowindows software (Modular Instruments).

The entire procedure for discrete microinjections into the subpostremal NTS has been described previously (3-7, 15, 25, 27, 28). Briefly, the animals were mounted in a cranial stereotaxic apparatus. The dorsal medulla was exposed at the level of the obex after dissection of the neck muscles and the atlanto-occipital membrane. Animals were allowed to stabilize for at least 30 min before microinjection of CGS-21680. Eight animals received the beta -adrenergic antagonist propranolol (2 mg/kg iv) ~10 min before microinjection. Unilateral microinjections of CGS-21680 were performed using multibarrel glass micropipettes (15- to 20-µm tip diameter for each barrel) into the middle to caudal one-third of the subpostremal NTS via a pneumatic pico-pump (model PV820, World Precision Instruments). A total volume of 50 nl was injected over 5-10 s. Because the pipette was pointed rostrally at the angle of 22° from the vertical plane and the rat skull was tilted 45°, nose down, the tip of the pipette reached the NTS 0.2-0.3 mm rostrally from the point of penetrating the surface of the brain stem. The surface coordinates for insertion of the micropipette relative to the caudal tip of the area postrema were as follows: anteriorposterior, -0.1 mm; mediolateral, 0.3 mm; and dorsoventral, 0.35 mm from the dorsal surface of the brain stem.

Previous studies (3, 6) have shown that CGS-21680 exhibits a steep dose-response curve for elicited hypotension. In the previous study measuring regional vascular response patterns (5), the approximate threshold dose (2 pmol in 50-nl volume) and the maximally effective hypotensive dose (20 pmol in 50-nl volume) were used. In this study, we were investigating the mechanisms producing the hindlimb vasodilation, so only the maximally effective hypotensive dose (20 pmol in 50-nl volume) was used. We chose this dose to maximally express all possible mechanisms participating in this marked hindlimb vasodilation.

The carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate, Molecular Probes; 0.1% solution in DMSO) was delivered from a separate barrel of the micropipette to mark the injection site for histological analysis. At the completion of the experiments, the animals were perfused transcardially with a 10% buffered Formalin and subsequently processed histologically in 64-µm coronal sections. These unstained tissue sections were examined via fluorescence microscopy to determine the site of injection marked by the DiI lipophilic dye. The injection sites were plotted on schematic representations of coronal sections of the rat subpostremal NTS (see Fig. 1) according to the atlas of Barraco et al. (2).


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  		Fig. 1.   Microinjection sites in subpostremal nucleus tractus solitarii (NTS) for all experiments. Two left panels show sites for the intact control and beta -adrenergic blockade. Two right panels show sites for lumbar sympathectomy, adrenalectomy, and combined lumbar sympathectomy and adrenalectomy. Schematic diagrams are transverse sections of medulla oblongata from a rat brain. NTS is shown at level of caudal portion of area postrema (AP). C, central canal; 10, dorsal motor nucleus of vagus nerve; 12, nucleus of hypoglossal nerve; Gr, gracile nucleus; Cu, cuneate nucleus. Number on left side of schematic diagram denotes rostrocaudal position of section (in mm) relative to obex according to an atlas of rat subpostremal NTS (2).

Data analysis. Iliac vascular conductance (IVC) was calculated by dividing iliac blood flow (IBF), expressed as a Doppler shift (Hz), by MAP. The units for conductance are Hz per millimeter of Hg. Responses for MAP, HR, IBF, and IVC were quantified in two ways: 1) maximal change compared with a 60-s basal control period immediately before microinjection, and 2) integration of the response over the period of change in the MAP (integral response). One-way ANOVA for independent measures was used to determine statistical significance. Differences were further evaluated by Fisher's least significant differences test. An alpha -level of P < 0.05 was used to determine significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The basal levels of hemodynamic parameters for each experimental group are shown in Table 1. The basal MAP value after bilateral sympathectomy was significantly lower than that seen in the intact, control animals. There was also a significant increase in both the basal IBF and IVC values following lumbar sympathectomy. beta -Adrenergic blockade significantly reduced the basal HR. Although there was a tendency for the basal MAP values to be lower after beta -adrenergic blockade, adrenalectomy, and combined lumbar sympathectomy and adrenalectomy, there was no statistically significant difference from the intact rats.

                              
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  		Table 1.   Resting values of hemodynamic parameters for each experimental group

Figure 2 shows a tracing from an intact animal, after beta -adrenergic blockade, and after combined sympathectomy and adrenalectomy. In the intact animals, microinjections of CGS-21680 (20 pmol/50 nl) into the subpostremal NTS elicited decreases in MAP and HR and increases in IBF and IVC consistent with previous studies (5, 27, 28). beta -Adrenergic blockade slightly reversed the MAP and IVC responses, and the combined sympathectomy and adrenalectomy virtually abolished the IVC response.


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  		Fig. 2.   Mean arterial pressure (MAP), heart rate (HR), iliac blood flow (IBF), and iliac vascular conductance (IVC) responses to microinjection of CGS-21680 (20 pmol in 50 nl) in intact animals, after beta -adrenergic blockade with propranolol (2 mg/kg iv), and after bilateral lumbar sympathectomy combined with adrenalectomy. Vertical arrows indicate microinjections. beta -Adrenergic blockade reversed MAP and IVC responses, and combined lumbar sympathectomy and adrenalectomy virtually abolished IVC response.

Figures 3 and 4 show the maximal responses and the integrals for MAP, HR, IBF, and IVC after stimulation of NTS adenosine A2a receptors. In the intact animal, MAP decreased an average of 26.6 ± 2.1% (26.7 ± 3.4 mmHg) from the basal control level, and HR decreased by 12.0 ± 1.4% (46.5 ± 5.7 beats/min). On average, NTS A2a purinoceptor activation increased IBF by 60.2 ± 12.6% (429 ± 115 Hz), and IVC increased by 110.2 ± 18.4% (8.2 ± 2.0 Hz/mmHg).


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  		Fig. 3.   Average maximum responses (top panels) and integral responses (bottom panels) of MAP (change in MAP, Delta mmHg) and HR (change in HR, Delta beats/min) evoked by microinjections of CGS-21680 (20 pmol in 50 nl) into the subpostremal NTS in intact animals, after beta -adrenergic blockade, after lumbar sympathectomy, after adrenalectomy, and after combined sympathectomy and adrenalectomy. *P < 0.05 vs. intact animals.



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  		Fig. 4.   Average maximum responses (top panels) and integral responses (bottom panels) of IBF (change in frequency, Delta Hz) and IVC (change in IVC, Delta Hz/mmHg) evoked by microinjections of CGS-21680 (20 pmol in 50 nl) into the subpostremal NTS in intact animals, after beta -adrenergic blockade, after lumbar sympathectomy, after adrenalectomy, and after combined sympathectomy and adrenalectomy. *P < 0.05 vs. intact animals; #P < 0.05 vs. combined adrenalectomy and lumbar sympathectomy.

beta -Adrenergic blockade reversed the MAP, HR, IBF, and IVC responses to stimulation of adenosine A2a receptors. Integral changes in HR were not different from zero. Instead of the typical decrease in MAP seen in the intact animal after microinjection of CGS-21680 into the subpostremal NTS, after pretreatment with propranolol, stimulation of adenosine A2a receptors increased MAP an average of 11.4 ± 1.5% (9.4 ± 1.5 mmHg). This increase was statistically significant. Activation of NTS adenosine A2a receptors after beta -adrenergic blockade produced a vasoconstriction in the iliac artery (Figs. 2 and 4), in contrast to the large vasodilation seen in the intact animal. These data suggest that a beta -adrenergic mechanism plays a significant role in mediating both the cardiac and peripheral effects of NTS adenosine A2a receptor activation.

Lumbar sympathectomy attenuated the maximal MAP response to NTS adenosine A2a receptor stimulation and did not significantly alter the HR responses. Both the maximal and integral IBF responses to adenosine A2a stimulation were reversed by bilateral lumbar sympathectomy, in comparison with the intact, control group. Lumbar sympathectomy significantly attenuated the IVC response to microinjection of CGS-21680 (Fig. 4). This indicates that the efferent sympathetic nerves play a role in the vasodilation evoked by adenosine A2a receptor stimulation.

Bilateral adrenalectomy also produced significant changes in the MAP, IBF, and IVC responses to NTS adenosine A2a receptor stimulation. Adrenalectomy had no significant effect on the HR response compared with the intact animals. The maximal change in MAP evoked by microinjection of CGS-21680 was significantly reduced following adrenalectomy. The large increase in IVC seen in the intact animals was also markedly attenuated in adrenalectomized animals. The IVC response produced by NTS adenosine A2a receptor activation in the intact animal was reduced by ~80% after adrenalectomy. This indicates that the adrenal glands play a substantial role in eliciting the vasodilation produced by stimulation of adenosine A2a receptors in the subpostremal NTS.

Combined lumbar sympathectomy and adrenalectomy attenuated the maximal decrease in MAP evoked by microinjection of CGS-21680 into the subpostremal NTS and had no significant effect on the HR response. The large preferential hindlimb vasodilation elicited by activation of adenosine A2a receptors in the subpostremal NTS was virtually abolished by removing both the lumbar sympathetic nerves and the adrenal glands. Residual changes in IVC following adenosine A2a receptor stimulation were not significantly different from zero in this group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study to directly examine the mechanisms mediating the preferential hindlimb vasodilation evoked by selective activation of adenosine A2a receptors in the subpostremal NTS. The major finding was that this vasodilation is mediated by both neural and humoral mechanisms. Bilateral removal of the adrenal glands or the efferent sympathetic nerves directed to hindlimbs markedly attenuated the vasodilatory response, whereas removal of both effector mechanisms (the sympathetic nerves and the adrenal glands) virtually abolished the response. Importantly, systemic blockade of beta -adrenergic receptors reversed hindlimb vasodilation and MAP depression elicited by stimulation of NTS A2a adenosine receptors, indicating that a beta -adrenergic mechanism plays a major role in mediating these responses.

Peripheral mechanisms triggered by NTS adenosine A2a receptors. The present study helps to explain our former "paradoxical" observation that stimulation of adenosine A2a receptors in subpostremal NTS evoked preferential hindlimb vasodilation; however, LSNA directed to the hindlimb vascular bed did not change in this setting (5, 27). There were two theoretical explanations for this "paradox": 1) vasodilation could be mediated by withdrawal of sympathetic vasoconstrictor activity and simultaneous increase in activity of vasodilatory fibers directed to the hindlimb so that the net level of efferent sympathetic activity did not change; and, less likely, 2) LSNA was not involved in this vasodilatory response, but the entire vasodilation was mediated via a humoral mechanism. The present data showed that lumbar sympathetic nerves are responsible for ~40% of hindlimb vasodilation elicited by stimulation of NTS A2a receptors. In addition, the baseline MAP decreased whereas IBF and IVC markedly increased following lumbar sympathectomy, indicating the presence of substantial, resting vasoconstrictor tone directed to the hindlimb. Because vasoconstrictor tone is a part of resting LSNA and because changes in LSNA contribute substantially to the marked hindlimb vasodilation evoked by stimulation of NTS A2a receptors, and given that we previously observed that LSNA did not change following A2a receptor stimulation, we then conclude that this vasodilation must be mediated by both withdrawal of vasoconstrictor tone and simultaneous activation of efferent vasodilatory fibers. Further studies using single fiber recordings of LSNA are required to confirm this conclusion. The specific mechanism mediating active sympathetic vasodilation in this setting remains unknown. However, two possibilities should be considered: 1) release of preabsorbed epinephrine from sympathetic terminals directed to the hindlimb vascular bed, according to the concept of Berecek and Brody (8; see also Ref. 14); and/or 2) active sympathetic vasodilation mediated via release of nitric oxide (11, 12, 24). Our observation that a beta -adrenergic mechanism plays a major role in this vasodilation suggests that the first possibility may be more likely.

Our data indicate that in addition to sympathetic withdrawal and active sympathetic vasodilation, a humoral mechanism, i.e., epinephrine released from the adrenal gland, is involved in mediating the preferential hindlimb vasodilation as was suggested by our previous study (28). Previously we have shown (27, 28) that stimulation of NTS A2a receptors markedly and selectively increased pre-ASNA, directed to the adrenal medulla, whereas it inhibited other postganglionic sympathetic outputs directed to adrenal and renal vasculatures. An increase in adrenal sympathetic nerve activity results in the release of epinephrine and norepinephrine from the adrenal medulla into the blood stream in a relative ratio of ~4:1 (17). Circulating epinephrine may contribute to the hindlimb vasodilation by its action on beta -adrenergic receptors, which are preferentially located in muscle vasculature (34). Therefore, it is likely that marked hindlimb vasodilation evoked by stimulation of NTS A2a receptors is mediated in part by epinephrine released from the adrenal glands, inasmuch as bilateral adrenalectomy abolished ~80% of this vasodilatory response and no vasodilation occurred after beta -adrenergic blockade.

Combined adrenalectomy and sympathectomy virtually abolished the vasodilation evoked by NTS adenosine A2a receptor activation. However, the adrenalectomy and lumbar sympathectomy effects on the IVC response were not additive, i.e., lumbar sympathectomy reduced ~40% of the vasodilatory response whereas adrenalectomy removed ~80% of the response. This suggests that the interaction between these two mechanisms is nonlinear. This interaction also supports the possibility that sympathetic terminals located in hindlimb vasculature took up and rereleased the epinephrine originating from the adrenal gland, as has been shown for hindlimb vasodilation evoked by stimulation of the anteroventral region of the third ventricle (AV3V) (8) or nicotine-induced dilation of muscle vascular bed of the hindlimb (14). Future studies using neuronal uptake inhibitors are required to determine the precise role of this mechanism in the vasodilation produced by NTS adenosine A2a receptor stimulation. There is still a significant decrease in MAP after elimination of the vasodilatory response by the combined removal of adrenal glands and lumbar sympathetic trunks. This could be due to changes in cardiac output or it could be a result of vasodilation in other vascular beds that were not denervated.

Systemic beta -adrenergic blockade reversed maximal MAP, IBF, IVC, and HR responses to stimulation of adenosine A2a receptors in the NTS and abolished the integral HR response. This indicates that the bradycardia produced by A2a purinoceptor activation is mediated primarily via withdrawal of tonic sympathetic activity. These data also strongly support the importance of both adrenal and neural mechanisms, possibly utilizing epinephrine to mediate hindlimb vasodilation in this setting. The vasoconstriction seen after stimulation of adenosine A2a receptors in animals under the conditions of beta -adrenergic blockade could be the result of alpha -adrenergic receptor action of epinephrine and norepinephrine released from the adrenal glands and/or sympathetic terminals. This mechanism may also explain the increase in MAP after stimulation of adenosine receptors in animals pretreated with propranolol. Although propranolol can cross the blood-brain barrier, as do most beta -adrenergic antgonists, we believe that the major effect of intravenously administered propranolol on the hindlimb vasodilatory response to stimulation of NTS A2a receptors was exerted via its peripheral action on vascular beta -receptors given that 1) activation of NTS A2a receptors causes marked increases in pre-ASNA (28); 2) the hindlimb vasodilatory response was markedly attenuated by adrenalectomy; and 3) the hindlimb vasodilatory response was abolished by combined adrenalectomy and sympathectomy.

Perspectives: central mechanisms triggered by NTS A2a receptors. Several lines of evidence strongly suggest that adenosine, operating via A2a receptors, facilitates baroreflex mechanisms at the level of the NTS. The hypotensive action of adenosine microinjected into the NTS is mediated via A2a receptors (3). Presynaptic A2a receptors are present on afferent vagal terminals in the NTS (9), and these receptors are known to facilitate the release of glutamate, a primary neurotransmitter in NTS baroreflex mechanisms (10, 18), from neural terminals (9, 20). In addition, the blockade of ionotropic glutamatergic mechanisms in the NTS attenuates hypotensive responses to microinjection of adenosine, and nonselective blockade of adenosine receptors attenuates HR baroreflex responses to increases in MAP (21). However, this mechanism alone cannot explain the results of our present and previous studies. For example, although stimulation of NTS A2a receptors decreases MAP, HR, RSNA, and post-ASNA, which is consistent with facilitation of the baroreflex mechanism, such stimulation increases pre-ASNA and does not change LSNA, whereas all these sympathetic outputs are uniformly inhibited by stimulation of peripheral baroreceptors or microinjection of glutamate into the NTS (26, 28). Taken together, our results and aforementioned reports by others implicate that the stimulation of NTS A2a receptors, in addition to facilitation of the NTS baroreflex mechanisms, triggers other mechanisms that selectively counteract regional baroreflex responses. For example, it is possible that the increase in pre-ASNA may be a result of selective inhibition of powerful, tonic baroreflex restraint of this sympathetic output (26). In support of this concept, presynaptic A2a receptors may facilitate the release of GABA in central structures (22), and GABA-ergic mechanisms inhibit baroreflex neurons at the level of the NTS during the hypothalamic defense response (19, 29). Interestingly, during the defense response triggered by experimental or natural factors (stressors) a marked, preferential hindlimb vasodilation occurs (11, 16, 36), similar to the effect evoked by stimulation of NTS A2a receptors (5). Both the defense response and stimulation of NTS A2a receptors activate the adrenal medulla, stimulate release of epinephrine, and evoke preferential hindlimb vasodilation at least in part via this humoral beta -adrenergic mechanism. Although during the defense response MAP and HR increase, these components of the response are facilitated by central adenosine A1 receptors (13, 30), whereas hindlimb vasodilation may be mediated by selective activation of NTS A2a receptors.

The lack of changes in LSNA, contrasting with the preferential hindlimb vasodilation following stimulation of NTS A2a receptors (5, 27), may be explained by simultaneous facilitation of two independent NTS mechanisms that control tonically active sympathetic vasoconstrictor fibers and phasically active sympathetic vasodilatory fibers contributing to LSNA and directed to the hindlimb vascular bed. It is possible that stimulation of NTS A2a receptors inhibits the tonic vasoconstrictor component of LSNA via facilitation of glutamatergic baroreflex mechanisms (9, 20, 21, 26). This decrease in LSNA is probably compensated by the simultaneous increase of an active vasodilatory component of LSNA (8, 11, 12, 24). This process may be triggered by A2a receptor-mediated stimulation of NTS glutamatergic neurons, which finally activate sympathetic vasodilatory fibers directed to the hindlimb, for example, via direct, glutamatergic, stimulatory connections between the NTS and rostral ventrolateral medulla (10). According to this hypothesis, stimulation of NTS A2a receptors may selectively activate two distinct NTS mechanisms: 1) it may facilitate an NTS baroreflex mechanism leading to withdrawal of vasoconstrictor tone, which is mediated via a peripheral alpha -adrenergic mechanism; and 2) it may trigger active hindlimb vasodilation via a beta -adrenergic mechanism consisting of both humoral and neural components. Both these mechanisms may support each other in creating the hypotension and preferential hindlimb vasodilation following stimulation of NTS A2a adenosine receptors.

In conclusion, the marked hindlimb vasodilation elicited by activation of adenosine A2a receptors in the subpostremal NTS is mediated by both the efferent sympathetic nerves and the adrenal glands. A beta -adrenergic mechanism plays a significant role in this response, suggesting that epinephrine released from the adrenal gland and possibly from the sympathetic terminals is involved in these marked hemodynamic responses.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance of C. Cupps. This study was supported by National Institutes of Health Grants MH-47181 and GM-08167.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. S. O'Leary, Dept. of Physiology, Wayne State Univ. School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201 (E-mail: doleary{at}med.wayne.edu).

Received 27 May 1999; accepted in final form 15 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 278(6):H1775-H1782
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