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Am J Physiol Heart Circ Physiol 278: H2057-H2068, 2000;
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Vol. 278, Issue 6, H2057-H2068, June 2000

Differential role of ionotropic glutamatergic mechanisms in responses to NTS P2x and A2a receptor stimulation

Tadeusz J. Scislo and Donal S. O'Leary

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of ATP P2x receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of alpha ,beta -methylene ATP (alpha ,beta -MeATP) elicits fast initial depressor and sympathoinhibitory responses that are followed by slow, long-lasting inhibitory effects. Activation of NTS adenosine A2a receptors via microinjection of CGS-21680 elicits slow, long-lasting decreases in arterial pressure and renal sympathetic nerve activity (RSNA) and an increase in preganglionic adrenal sympathetic nerve activity (pre-ASNA). Both P2x and A2a receptors may operate via modulation of glutamate release from central neurons. We investigated whether intact glutamatergic transmission is necessary to mediate the responses to NTS P2x and A2a receptor stimulation. The hemodynamic and neural (RSNA and pre-ASNA) responses to microinjections of alpha ,beta -MeATP (25 pmol/50 nl) and CGS-21680 (20 pmol/50 nl) were compared before and after pretreatment with kynurenate sodium (KYN; 4.4 nmol/100 nl) in chloralose-urethan-anesthetized male Sprague-Dawley rats. KYN virtually abolished the fast responses to alpha ,beta -MeATP and tended to enhance the slow component of the neural responses. The depressor responses to CGS-21680 were mostly preserved after pretreatment with KYN, although the increase in pre-ASNA was reduced by one-half following the glutamatergic blockade. We conclude that the fast responses to stimulation of NTS P2x receptors are mediated via glutamatergic ionotropic mechanisms, whereas the slow responses to stimulation of NTS P2x and A2a receptors are mediated mostly via other neuromodulatory mechanisms.

nucleus tractus solitarii; purinergic receptors; ionotropic glutamatergic blockade; renal sympathetic nerve; adrenal sympathetic nerve


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BOTH ATP AND ADENOSINE PLAY an important role in processing cardiovascular control at the level of the nucleus tractus solitarii (NTS) (1, 4, 6, 7, 13, 22, 24, 25, 27, 31, 33-35, 41). For example, blockade of P2x receptors in the subpostremal NTS severely impairs baroreflex control of heart rate, suggesting an important physiological role for these receptors in NTS baroreflex mechanisms (33). Recently, it was reported that ATP, a natural agonist of P2x receptors, is neuronally released into the NTS during activation of the hypothalamic defense response (39). Released ATP is subsequently catabolized by 5'-ectonucleotidase to adenosine, which acts further as a neuromodulator in this response. Adenosine may be also naturally released into the NTS as a result of intracellular breakdown of ATP during hypoxia, ischemia, and severe hemorrhage (28, 42, 44). Extracellular ATP may act via postsynaptic P2x receptors as an independent, fast neurotransmitter between central neurons or as a cotransmitter with glutamate (11, 12, 20). ATP may also facilitate the release of glutamate from afferent synaptic terminals via presynaptic P2x receptors (16, 21). Adenosine operating via presynaptic A2a receptors may facilitate synaptic release of different neurotransmitters/neuromodulators, including glutamate, into the NTS (3, 5, 7, 24). Because both ATP and adenosine interact with central glutamatergic mechanisms, and because glutamate operates as a primary, fast neurotransmitter in baro- and chemoreflex pathways at the level of the NTS (22), possible interaction between purinergic and glutamatergic mechanisms in the NTS may play an important role in central cardiovascular responses to different physiological and pathological situations.

Our recent studies (6, 31, 35) showed that activation of P2x receptors in the caudal subpostremal NTS via microinjection of selective agonist alpha ,beta -methylene ATP (alpha ,beta -MeATP) elicits dose-dependent decreases in mean arterial pressure (MAP) and heart rate (HR) and differential inhibition of regional sympathetic nerve activity. These responses consist of fast (neuromediator- like) and slow (neuromodulator-like) components. The fast (seconds) component of the responses to stimulation of NTS P2x receptors mimics hemodynamic and differential neural responses to stimulation of arterial baroreceptors as well as those responses to direct microinjection of glutamate into the NTS (32, 33). The slow component is qualitatively similar to the fast one, but it has a much smaller amplitude and lasts markedly longer (minutes). The striking similarities between the fast cardiovascular and neural responses to P2x and those to glutamatergic receptor stimulation indicate that this part of the response is likely mediated via activating glutamatergic mechanisms, whereas the slow, long-lasting response to P2x receptor stimulation may be mediated via other slow-acting neuromodulatory mechanisms.

We also recently showed (6, 34, 35) that activation of adenosine A2a receptors in the subpostremal NTS elicits slow, long-lasting responses that include decreases in MAP, HR, renal sympathetic nerve activity (RSNA), and postganglionic adrenal sympathetic nerve activity. However, preganglionic adrenal nerve activity (pre-ASNA) markedly increases in this setting (35). Because stimulation of glutamatergic receptors in the same site of the NTS, as well as maximal activation of arterial baroreceptors, inhibits all of the aforementioned regional sympathetic outputs (32, 35), the effects of stimulation of NTS A2a receptors must be mediated, at least in part, via nonglutamatergic mechanisms. Our recent observations (7, 24, 25) contrast with the concept that adenosine acts in the NTS mostly via presynaptic facilitation of glutamate release. That hypothesis was based on observations that adenosine enhanced the release of glutamate into the NTS, nonselective blockade of adenosine receptors in the NTS attenuated baroreflex responses, and blockade of glutamatergic transmission in the NTS attenuated depressor action of adenosine microinjected into the NTS (7, 24, 25). However, the effect of blockade of ionotropic glutamatergic receptors on selective stimulation of NTS A2a adenosine receptors has not been studied.

Because the interaction between NTS purinoceptor action and glutamatergic transmission remains unclear, our present study was designed to evaluate the contribution of NTS ionotropic glutamatergic mechanisms to the responses evoked by selective stimulation of purinergic P2x and A2a receptors. To stimulate NTS purinoceptor subtypes and record differential hemodynamic and neural responses, we used the same experimental design used in our previous studies (31, 34, 35). We hypothesized that blockade of ionotropic glutamatergic receptors in subpostremal NTS will attenuate the fast but not the slow response to P2x receptor stimulation and that the response to stimulation of A2a receptors is partially independent of the fast glutamatergic transmission within the NTS. Our results confirmed this hypothesis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND 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 effect of blockade of glutamatergic ionotropic receptors or respective volume control on the responses to stimulation of P2x and A2a purinergic receptors in the subpostremal NTS was studied in 38 male Sprague-Dawley rats (350-400 g) (Charles River). In nine animals the responses to stimulation of NTS P2x receptors with microinjections of the selective agonist alpha ,beta -MeATP were compared before and after microinjection of kynurenate sodium (KYN) into the same site of the NTS. In seven animals the responses to alpha ,beta -MeATP were compared before and after respective volume control (artificial cerebrospinal fluid, ACF). NTS A2a-adenosine receptors were stimulated with the selective agonist CGS-21680 after pretreatment with KYN in 10 animals and after the respective volume control (ACF) in 7 animals. In two additional groups of animals (n = 5 for each group) the time control for all recorded parameters was evaluated after microinjection of KYN alone, and the efficacy of the blockade of NTS glutamatergic receptors to impair baroreflex responses was assessed.

Instrumentation and measurements. All the procedures were described in detail previously (4, 6, 13, 31, 34, 35). Briefly, rats were anesthetized with a mixture of alpha -chloralose (80 mg/kg) and urethan (500 mg/kg ip), tracheotomized, and artificially ventilated with oxygen-enriched air. Arterial blood gases were tested occasionally (ABL500, OSM3; Radiometer), and ventilation was adjusted to maintain PO2, PCO2, and pH within normal ranges. The right femoral artery and vein were catheterized to monitor arterial blood pressure and infuse drugs.

The adrenal and renal nerves were exposed retroperitoneally, and neural recordings were accomplished as described previously (31, 32, 34, 35). Neural signals were initially amplified (×2,000-20,000) with bandwidth set at 100-1,000 Hz and then digitized, rectified, and averaged in 1-s intervals. Background noise was determined 30-60 min after the animal was euthanized. Resting nerve activity before any intervention was normalized to 100%.

The ratio of preganglionic to total nerve activity was initially tested with bolus injection of the short-lasting (1-2 min) ganglionic blocker arfonad (2 mg/kg) (32, 35) and was reevaluated at the end of each experiment with hexamethonium (20 mg/kg iv). According to our previous criteria, pre-ASNA was considered as predominantly preganglionic if the activity remaining after ganglionic blockade at the end of each experiment was >75% (35). After final ganglionic blockade, average pre-ASNA and RSNA were 109.4 ± 3.5% and 3.6 ± 0.6%, respectively. The increase of pre-ASNA over 100% was likely due to an arterial baroreflex response caused by the decrease in MAP after ganglionic blockade.

The arterial pressure and neural signals were digitized and recorded with a Hemodynamic and Neural Data Analyzer (Biotech Products, Greenwood, IN), averaged over 1-s intervals, and stored on a hard disk for subsequent analysis.

Microinjections into the NTS. Unilateral microinjections of alpha ,beta -MeATP, CGS-21680, KYN, vehicle (ACF), and carbocyanine dye (DiI) were made with three-barrel glass micropipettes into the medial region of the caudal subpostremal NTS as described previously (4, 6, 31, 33-35). Briefly, with the rat skull adjusted to a 45° angle from the horizontal plane of the stereotaxic apparatus and with the micropipette barrel held at a 22° angle from the vertical plane, the surface coordinates used for insertion of the micropipette relative to the caudal tip of the area postrema were as follows: anteroposterior = -0.1 mm, mediolateral = 0.3 mm, and dorsoventral = 0.35 mm from the dorsal surface of the brain stem. All the drugs were dissolved in ACF, and the pH was adjusted to 7.2. No more than one microinjection protocol was performed on one side of the NTS. All microinjection sites were verified histologically as described previously (31, 33-35) and presented schematically in Fig. 1.


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  		Fig. 1.   Microinjection sites in subpostremal nucleus tractus solitarii (NTS) for all experiments. Schematic diagrams show transverse sections of medulla oblongata from a rat brain. AP, area postrema; c, central canal; 10, dorsal motor nucleus of vagus nerve; 12, nucleus of hypoglossal nerve; Ts, tractus solitarius; Gr, gracile nucleus; Cu, cuneate nucleus. Numbers on left denote rostrocaudal position (in mm) of section relative to obex according to the atlas of rat subpostremal NTS by Barraco et al. (2). Microinjection sites were marked with fluorescent dye and are denoted by symbols: , alpha ,beta -methylene ATP (alpha ,beta -MeATP) before and after blockade of glutamatergic receptors; open circle , alpha ,beta -MeATP before and after control microinjection of 100 nl of artificial cerebrospinal fluid (ACF); black-triangle, CGS-21680 after blockade of glutamatergic receptors; triangle , CGS-21680 after control microinjection of 100 nl of ACF; , bilateral microinjections of kynurenate sodium (KYN, 4.4 nmol/100 nl) for evaluation of time course of glutamatergic receptor blockade; , unilateral microinjections of KYN (4.4 nmol/100 nl) for evaluation of spontaneous changes in resting values after blockade.

Our previous studies (4, 6, 13, 31, 34, 35) showed that microinjections of selective P2x and A2a receptor agonists, alpha ,beta -MeATP and CGS-21680, respectively, into the subpostremal NTS evoke dose-related responses in all hemodynamic and neural parameters recorded in the present study. We also showed previously (13, 31) that the effects elicited with the highest dose investigated of both purinoceptor agonists were completely and selectively blocked by microinjection of the selective P2 purinoceptor antagonist suramin or the A2a purinoceptor antagonist CGS-15943A (4), respectively. In the present study a relatively low dose, 25 pmol/50 nl of alpha ,beta -MeATP, was used. This dose of alpha ,beta -MeATP evoked a fast response that was very similar to the response evoked by microinjection of glutamate (100 pmol/50 nl) into the same site of the NTS that we showed in our previous study (35); the responses had a similar time course, magnitude, and relative ratio of differential inhibition of regional sympathetic outputs (35). Therefore, we used this particular dose of alpha ,beta -MeATP to evaluate possible interactions between NTS P2x and glutamatergic mechanisms. In addition, with this dose of the P2x receptor agonist, the fast and slow responses were well distinguished.

Responses to stimulation of NTS A2a receptors were expected to be mediated mostly via release of glutamate from neural terminals located in the NTS (7, 24). However, other mechanisms could also participate (37), e.g., release of norepinephrine or serotonin on stimulation of NTS A2a receptors (3, 5). Therefore, we used the maximal effective dose of the selective A2a receptor agonist CGS-21680 (20 pmol/50 nl) to maximally activate all possible mechanisms triggered by A2a receptor stimulation and to evaluate the contribution of glutamatergic mechanisms to this response.

Ionotropic glutamatergic blockade. The blockade of glutamatergic ionotropic receptors was accomplished with unilateral microinjection of KYN (4.4 nmol/100 nl), a dose and volume that completely block baroreflex responses when microinjected bilaterally (17, 40). The effectiveness of the glutamatergic receptor blockade was assessed in a separate group of animals (n = 5) on the basis of impairment of baroreflex responses after bilateral microinjection of the same dose and volume of KYN used in the main protocols. Baroreflex responses were evoked by ramp increases in MAP via slow intravenous infusion of phenylephrine (3.3 µg in 17 µl over 20 s) before and at different times after the microinjection of KYN. After KYN was microinjected, the increases in MAP evoked by the same doses of phenylephrine were markedly enhanced, as expected, whereas baroreflex responses in HR, RSNA, and pre-ASNA were markedly attenuated (Table 1). A two-way ANOVA for repeated measures indicated that before blockade, baroreflex gain for RSNA was significantly greater than that for pre-ASNA (P = 0.011 and P = 0.029 for maximal and integral values, respectively) and that the gains for both sympathetic outputs were similarly attenuated following microinjection of KYN (nonsignificant time vs. output interaction: P = 0.619 and P = 0.931 for maximum and integral responses, respectively) (Fig. 2). These results are consistent with those of our previous study (32), in which greater baroreflex gain for RSNA than for pre-ASNA was reported on the basis of analysis of whole sigmoidal baroreflex response curves obtained for these sympathetic outputs under similar experimental conditions. This indicates that the limited assessment of baroreflex gain performed in the present study was physiologically relevant. Blockade of ionotropic glutamatergic receptors in the subpostremal NTS severely and reversibly impaired the HR baroreflex gain and baroreflex inhibition of efferent sympathetic nerve activity for >20 min (Table 1 and Fig. 2). Therefore, the analysis of the effects evoked by stimulation of the purinergic receptors in the NTS was restricted to 20 min following pretreatment with KYN or an equivalent volume of ACF.

                              
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  		Table 1.   Baroreflex responses of HR, RSNA, and pre-ASNA to increases in MAP



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  		Fig. 2.   Baroreflex gain evaluated by increases in mean arterial pressure (MAP) via slow intravenous infusion of phenylephrine (3.3 µg in 17 µl during 20 s) before and at different times after bilateral microinjection of KYN at same dose used in main protocol (4.4 nmol/100 nl, time 0; n = 5). Blockade of ionotropic glutamatergic receptors in subpostremal NTS severely and reversibly impaired heart rate (HR) and neural baroreflex responses for over 20 min. A: maximal responses; B: integral responses. RSNA, renal sympathetic nerve activity; ASNA, preganglionic adrenal sympathetic nerve activity; Delta bpm, change in HR (in beats/min); Delta mmHg, change in MAP.

Unilateral microinjection of KYN resulted in a slow, ramplike increase in MAP and neural activity and then stabilization of these parameters ~5 min after the microinjection (Table 2 and Fig. 3). Stimulation of P2x or A2a receptors was performed during this stable period. However, after several minutes of relatively stable responses to KYN, the hemodynamic and neural parameters started to return very slowly toward their resting values, and these spontaneous changes were superimposed on the long-lasting responses to stimulation of P2x or A2a receptors. Therefore, in a separate group of five animals a time course of the spontaneous changes following the microinjection of KYN was evaluated for all recorded parameters (n = 9) (Table 3), and these spontaneous changes were subtracted from the responses to stimulation of the purinergic receptors after KYN was microinjected. Short-lasting and relatively small responses to microinjection of vehicle (ACF, 100 nl), similar to random fluctuations in all recorded parameters, were not subtracted.

                              
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  		Table 2.   Changes in resting values of MAP, HR, RSNA, and pre-ASNA following unilateral blockade of glutamatergic receptors in subpostremal NTS (after KYN) and respective volume control (after ACF)



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  		Fig. 3.   Responses to stimulation of NTS P2x receptors before and after microinjection of vehicle (ACF; A) and before and after blockade of ionotropic glutamatergic receptors with KYN (B). Microinjections of ACF and KYN were made into same site of NTS in 2 different animals. Note that fast but not slow component of response to alpha ,beta -MeATP was abolished after blockade. Note also that slow responses in pre-ASNA were markedly smaller than those in RSNA (B) and were sometimes virtually absent (A).


                              
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  		Table 3.   Changes in resting values of MAP, HR, RSNA, and pre-ASNA from levels that stabilized 5 min after unilateral blockade of glutamatergic receptors in subpostremal NTS

Experimental protocols. Two different protocols were applied: 1) a longitudinal protocol for stimulation of P2x receptors located in the same site of the NTS before and after pretreatment with KYN or ACF, and 2) a parallel protocol for stimulation of A2a receptors after pretreatment with KYN or ACF in different sides of the NTS or in different animals. The longitudinal protocol started with a control microinjection of alpha ,beta -MeATP, followed by a 30-min interval, microinjection of KYN, a 5-min interval, and, finally, a test microinjection of alpha ,beta -MeATP. The same protocol was repeated with an equivalent volume of ACF instead of KYN. The longitudinal design was extremely difficult to apply to the microinjections of CGS-21680 because this drug often precipitates within the micropipette on longer contact with tissue. Therefore, we used a parallel design that started with microinjection of KYN or ACF, followed by a 5-min interval and then microinjection of CGS-21680. The microinjections of KYN + CGS-21680 or ACF + CGS-21680 were made either in different animals or into different sides of the NTS in one animal with at least a 90-min interval between the injections.

Data analysis. Hemodynamic and sympathetic nerve responses were quantified in two ways as described previously (6, 31, 34, 35): 1) the maximal percent difference from a 30-s basal control period that was taken immediately before microinjection, and 2) integration of percent changes from control over the period of change in MAP but no longer than 20 min (time of effective glutamatergic blockade). The resting values (control period) were measured during the stable response to glutamatergic blockade, ~5 min after the microinjection of KYN or the respective ACF control. Slow, spontaneous changes (recovery) in all recorded parameters, following the stable response to glutamatergic blockade, were subtracted from both maximal and integral values of the responses to stimulation of P2x and A2a receptors (respective values are presented in Table 3). The HR responses, calculated from the pulse intervals, were expressed in absolute values (beats/min). Neural recordings were additionally filtered using a running average in 10-s intervals to minimize the effect of random spikes on maximum response values. Because the responses to alpha ,beta -MeATP exhibited a biphasic pattern, the fast and slow components of the responses were evaluated separately. The fast component was considered from the beginning of the response to the end of fast recovery, usually followed by a subsequent decline of all the parameters, which started the slow component (see Fig. 3). The slow component was evaluated over the remaining period of the decrease in MAP. Because the fast response was undetectable under the conditions of ionotropic glutamatergic blockade, the maximum and integral changes in all recorded parameters were evaluated for the average time period of the fast response measured before the blockade (Table 4). The effects of KYN and ACF on the resting hemodynamic and neural parameters were evaluated 5 min after the microinjection (Table 2). Baroreflex gain was expressed as change in HR and percent change in RSNA and pre-ASNA per change in MAP for both maximal and integral responses calculated over the period of induced increase in MAP. A one-way ANOVA for independent measures was used to compare MAP and HR responses, and a two-way ANOVA for independent measures was used to compare neural responses in parallel protocols (responses to CGS-21680 after ACF vs. after KYN). A one-way ANOVA for repeated measures was used to compare hemodynamic responses, and a two-way ANOVA for repeated measures was used to compare neural responses in longitudinal protocols (responses to alpha ,beta -MeATP before vs. after KYN and before vs. after ACF, and assessment of baroreflex responses before vs. after KYN). The differences were further evaluated by a modified Bonferroni t-test. The effects of KYN and ACF on the resting values were compared by the t-test for independent measures. An alpha  level of P < 0.05 was used to determine statistical significance.

                              
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  		Table 4.   Time to maximum responses and recovery of MAP after microinjections of alpha ,beta -MeATP or CGS-21680 before and after pretreatment with KYN or control microinjections of ACF


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The resting values for MAP and HR before microinjection of drugs or vehicle into the subpostremal NTS were 82.7 ± 1.4 mmHg and 360 ± 5 beats/min (n = 38). Unilateral blockade of ionotropic glutamatergic receptors in the subpostremal NTS increased MAP, decreased HR, and markedly increased pre-ASNA compared with a very small increase in RSNA (Table 2). Microinjection of the same volume of ACF did not markedly affect any of these parameters, although very small differences, smaller than the random fluctuations of these parameters that normally occur under resting conditions, were statistically significantly different from the control values that were averaged over the 30-s control period (Table 2).

P2x receptor stimulation. The effects of stimulation of NTS P2x receptors via microinjection of the selective agonist alpha ,beta -MeATP (25 pmol/50 nl) into the subpostremal NTS before and after pretreatment with KYN and ACF are presented in Fig. 3. The biphasic response to alpha ,beta -MeATP consists of a fast component, lasting ~30 s, followed by a slow, long-lasting component of the response (35). Blockade of ionotropic glutamatergic receptors in the NTS abolished the fast response but did not attenuate the slow response to alpha ,beta -MeATP (Fig. 3B). Microinjection of the same volume of ACF before microinjection of alpha ,beta -MeATP did not markedly affect either component of the response (Fig. 3A). This tendency was confirmed by analyses of averaged data, which are presented in Figs. 4 and 5 and Table 5. The fast neural and hemodynamic responses to alpha ,beta -MeATP were virtually abolished by blockade of ionotropic glutamatergic receptors located in the same site of the NTS, especially so for the integral responses. The residual decrements observed in all parameters after the blockade were not different from absolute values of random changes in these parameters during the time of the measurements, and therefore they reflect only natural variability. In contrast, the slow neural responses to stimulation of NTS P2x purinoceptors tended to increase after the blockade of glutamatergic ionotropic receptors; however, these increases did not reach statistical significance (Fig. 5, P = 0.077 and P = 0.117 for RSNA and pre-ASNA maximal responses, respectively). The glutamatergic blockade decreased the rate of development and decay of the slow responses to P2x receptor stimulation. Time to maximal depressor response and time to recovery were markedly prolonged after pretreatment with KYN (Table 4). Pretreatment with the same volume of ACF did not markedly affect the responses except for a small but significant decrease in the magnitude of fast MAP responses (Table 5). Although the time to maximum of the slow response increased after ACF, this increase was significantly smaller than that observed after KYN (Table 4). In three experiments, a gradual recovery of the fast response to microinjections of alpha ,beta -MeATP was also observed. Approximately 35 min after microinjection of KYN, the maximal fast responses to P2x receptor stimulation recovered to 34.3 + 8.6%, 42.0 + 6.6%, and 40.0 + 6.7% of control responses evoked before KYN for MAP, RSNA, and pre-ASNA, respectively. In one of these animals, also studied 75 min after KYN, the responses recovered further to 65%, 97%, and 92% for MAP, RSNA, and pre-ASNA, respectively.


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  		Fig. 4.   Average fast maximum responses (A) and integral responses (B) of MAP, HR, RSNA, and pre-ASNA evoked by microinjections of alpha ,beta -MeATP into caudal subpostremal NTS before (MeATP) and after ionotropic glutamatergic receptor blockade (KYN + MeATP) (n = 9). Blockade of ionotropic glutamatergic receptors virtually abolished hemodynamic and differential neural responses. *P < 0.05 vs. MeATP; #P < 0.05 vs. RSNA.



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  		Fig. 5.   Average slow maximum responses (A) and integral responses (B) of MAP, HR, RSNA, and pre-ASNA evoked by microinjections of alpha ,beta -MeATP into caudal subpostremal NTS before (MeATP) and after ionotropic glutamatergic receptor blockade (KYN + MeATP) (n = 9). Hemodynamic and differential neural responses were completely preserved after blockade. #P < 0.05 vs. RSNA.


                              
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  		Table 5.   Hemodynamic and neural responses to microinjections of alpha ,beta -MeATP into subpostremal NTS before and after pretreatment with microinjection of ACF

A2a receptor stimulation. The effect of blockade of glutamatergic ionotropic receptors and a respective volume control (ACF) on the responses to stimulation of NTS A2a receptors with the selective agonist CGS-21680 are presented in Fig. 6. The hemodynamic and differential neural responses to stimulation of NTS A2a receptors were similar to those reported previously (34, 35). The decreases in MAP, HR, and RSNA in response to A2a receptor stimulation were similar after pretreatment with KYN and ACF. Glutamatergic blockade markedly increased pre-ASNA, and further increases in pre-ASNA following the stimulation of A2a receptors were limited compared with those observed after pretreatment with ACF (Fig. 6). A two-way ANOVA indicated that the glutamatergic blockade had a significantly different impact on the responses from the two analyzed sympathetic outputs (drug vs. neural output interaction, P = 0.006 and P = 0.002 for maximum and integral responses, respectively). Averaged responses of pre-ASNA to stimulation of A2a receptors were markedly attenuated, especially for the integral responses (P < 0.05 for both maximum and integral values) (Fig. 7). The responses of all other parameters also tended to decrease as a result of the glutamatergic blockade (P = 0.085 and P = 0.20 for maximal and integral depressor responses, respectively); however, only the attenuation of the integral response in RSNA reached the level of statistical significance (P = 0.029) (Fig. 7).


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  		Fig. 6.   Responses to stimulation of NTS A2a receptors after pretreatment with ACF (100 nl; left) and after blockade of ionotropic glutamatergic receptors with KYN (4.4 nmol/100 nl; right) in same site of NTS. Recordings were made in 2 different animals. Blockade markedly increased pre-ASNA and limited its response to microinjection of CGS-21680.



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  		Fig. 7.   Average maximum responses (A) and integral responses (B) of MAP, HR, RSNA, and pre-ASNA evoked by microinjections of CGS-21680 into caudal subpostremal NTS after ionotropic glutamatergic receptor blockade (KYN + CGS-21680) (n = 9) and after respective volume control (ACF + CGS-21680) (n = 7). *P < 0.05 vs. ACF + CGS-21680; #P < 0.05 vs. RSNA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study to investigate the interaction between the effects of selective stimulation of purinergic receptor subtypes and glutamatergic ionotropic mechanisms in the NTS. The major new finding of the present study is that blockade of ionotropic glutamatergic receptors in the subpostremal NTS virtually abolished the fast but not the slow component of the response to stimulation of NTS P2x receptors. This indicates that the biphasic response to stimulation of NTS P2x receptors is mediated via at least two different mechanisms: glutamatergic for the fast component of the response and nonglutamatergic for the slow component of the response. We also found that hemodynamic and neuroinhibitory responses to the selective stimulation of adenosine A2a receptors were mostly preserved after the blockade of NTS ionotropic glutamatergic receptors, whereas the increase in ASNA was markedly attenuated after the blockade. This suggests that the major part of the responses to stimulation of NTS A2a receptors is mediated by nonionotropic glutamatergic mechanisms.

P2x receptors. The biphasic response to stimulation of NTS P2x receptors consists of fast and slow components, as we showed previously (35). The hemodynamic and neural patterns of the fast component of the response to a moderate dose of alpha ,beta -MeATP (25 pmol/50 nl) resembled very closely the time course and pattern of the response to microinjection of glutamate into the same site of the NTS (35). Therefore, we hypothesized that this part of the response is mediated via glutamatergic mechanisms. The present study confirmed this concept inasmuch as blockade of ionotropic glutamatergic receptors in the same site of the NTS virtually abolished the fast component of the response to P2x receptor stimulation. The nature of the interaction between NTS P2x and glutamatergic mechanisms remains unclear. Extracellular ATP may act via P2x receptors as an independent neurotransmitter between NTS interneurons linked in chain with glutamatergic neurons, similarly to the manner suggested for medial habenula or hippocampal slices (11, 12, 20), or ATP may be coreleased with glutamate and facilitate glutamatergic transmission as was described in the dorsal horn, trigeminal nucleus, and hippocampus (16, 20, 21). Further studies at the cellular level are required to elucidate this interaction.

In contrast, the slow component of the response to stimulation of NTS P2x receptors was not attenuated and even tended to be enhanced after the glutamatergic blockade. This indicated that NTS ionotropic glutamatergic mechanisms were not involved in mediating the slow part of the response. Because KYN impairs only the ionotropic glutamatergic mechanisms, it is theoretically possible that glutamatergic metabotropic receptors could be responsible for this response. However, it is unlikely because the responses to stimulation of glutamatergic metabotropic receptors are relatively short lasting (seconds) (15, 40), whereas the slow component of the response to alpha ,beta -MeATP lasted several minutes (see Table 4). The time course and amplitude of the slow hemodynamic and RSNA responses to P2x receptor stimulation resembled the responses to stimulation of A2a receptors (compare Figs. 5 and 7 and Table 5); however, stimulation of NTS P2x receptors decreased pre-ASNA, whereas stimulation of A2a receptors markedly increased pre-ASNA. Therefore, the slow component of the response to alpha ,beta -MeATP was not likely a result of possible nonspecific or indirect activation of A2a receptors. The other slow-acting neuromodulators, possibly triggered by stimulation of NTS P2x receptors, could be responsible for the observed long-lasting hemodynamic and neural responses. Considering that there exists a long list of NTS neuromodulators that may be directly or indirectly involved in this response, further detailed studies are necessary. Unfortunately, except for some data on general changes in MAP and HR, there is almost no information about differential regional neuroregulatory effects of the most neuroactive substances operating in the NTS (22), and this makes the search relatively difficult to focus at this time.

A2a receptors. Our present data indicate that hemodynamic and neural responses evoked by selective stimulation of NTS A2a receptors were, at most, only partially mediated via glutamatergic mechanisms. The major portion of the responses persisted after effective blockade of ionotropic glutamatergic receptors. These results remain in some contrast to the hypothesis that the hypotensive action of adenosine microinjected into the NTS is mediated via facilitating glutamate release from baroreceptor afferents terminating in the NTS (7, 24, 25). That concept was supported by the following observations: 1) blockade of ionotropic glutamatergic mechanisms in the NTS attenuated hypotensive responses to microinjection of adenosine (24), 2) nonselective blockade of adenosine receptors attenuated HR baroreflex responses to increases in MAP (25), and 3) glutamate was released into the NTS on microinjection of adenosine (24). It was previously reported that the hypotensive action of adenosine is mediated via A2a receptors (4), that A2a receptors facilitate the release of glutamate from neural terminals in central structures (7, 19, 29, 37), and that A2a receptors are present on presynaptic vagal terminals in the NTS (7). Therefore, it was tempting to conclude that hypotensive action of adenosine in the NTS is mediated mostly via stimulation of presynaptic A2a receptors and release of glutamate from afferent terminals and/or intrinsic NTS interneurons participating in the baroreflex mechanism. However, this hypothesis was never previously confirmed or denied in a direct way, i.e., with selective stimulation of A2a receptors before and after blockade of glutamatergic mechanisms in the NTS. In addition, the most recent studies from our laboratory showed that stimulation of NTS A2a receptors inhibits RSNA but markedly increases pre-ASNA, whereas stimulation of glutamatergic receptors in the same site of the NTS or stimulation of arterial baroreceptors powerfully inhibits both of these sympathetic outputs (32, 35). These observations strongly suggest that the effect of selective stimulation of NTS A2a receptors should be mediated, at least partially, by nonglutamatergic mechanisms. The present study confirmed this hypothesis, indicating that that the RSNA and hemodynamic effects of selective stimulation of A2a receptors were mostly preserved after blockade of glutamatergic receptors in the same site of the NTS.

The increase of pre-ASNA in response to A2a receptor stimulation was markedly attenuated after the glutamatergic blockade. However, the glutamatergic blockade increased (disinhibited) pre-ASNA compared with other variables (see Table 2). Therefore, the potential for the further increases in pre-ASNA after glutamatergic blockade may have been limited. The greater increase in pre-ASNA than RSNA following glutamatergic blockade is consistent with our previous observation that unloading of arterial baroreceptors under similar experimental conditions disinhibits pre-ASNA to a greater extent than RSNA and lumbar sympathetic nerve activity (32). Taken together, our present and previous observations suggest that the increase in pre-ASNA evoked by stimulation of NTS A2a receptors may be a result of selective disinhibition of baroreflex restraint directed to pre-ASNA. This disinhibition may be mediated via A2a receptor-triggered facilitation of intrinsic NTS GABA-ergic mechanisms, similarly to that observed during the hypothalamic defense response (23, 38). In support of this concept, a very recent study by Phillis (26) showed that stimulation of A2a receptors in the rat cortex may evoke inhibition of cortical neurons via a GABA-ergic mechanism. There is also a possibility that KYN may impair an A2a-receptor-triggered active glutamatergic stimulation of pre-ASNA via mechanisms not related to baroreflex control of this sympathetic output.

The specific mechanisms responsible for the effects of NTS A2a receptor stimulation under the condition of ionotropic glutamatergic blockade remain unknown. Activation of A2a receptors may facilitate the release of several different neurotransmitters/neuromodulators from central neurons (3, 5, 14, 26, 37). Therefore, even if basic glutamatergic transmission in the NTS is severely impaired, A2a receptor stimulation may still activate or disinhibit outgoing NTS neurons, as well as their glutamatergic terminals located in the ventrolateral medulla or nucleus ambiguus, distantly enough to be unaffected by the blockade. Among the numerous possibilities, norepinephrine and serotonin should be considered. Both of these substances are known to operate in the NTS and evoke long-lasting depressor responses on administration into the NTS (22), and both are released on stimulation of NTS A2a receptors in vitro (3, 5). Interestingly, serotonin is involved in the central processing of the responses to hemorrhagic shock and stimulation of cardiac chemoreceptors, and activation of both these mechanisms inhibits RSNA, whereas it enhances pre-ASNA similarly to that occurring during selective stimulation of NTS A2a receptors (18, 35, 36, 43).

It is still unclear why the depressor effect of adenosine microinjected into the NTS was significantly attenuated by KYN, as previously reported by Mosqueda-Garcia and colleagues (24), whereas in the present study the depressor effect of selective stimulation of NTS A2a-adenosine receptors was only slightly, nonsignificantly (P = 0.085) decreased after pretreatment with KYN. One possibility is that these authors used an exceptionally high dose of kynurenic acid (33 nmol/60 nl) and that even this high dose of the antagonist only partially attenuated the response to adenosine. Another possibility is that the interaction between glutamatergic mechanisms and adenosine versus selective stimulation of adenosine A2a receptors may be different. For example, adenosine acting simultaneously via at least three different receptor subtypes (A1, A2a, and A3) may facilitate and simultaneously inhibit the release of other neurotransmitters/neuromodulators with different ratios from that occurring during selective stimulation of A2a receptors (30).

Limitations of the method. A limitation of this study is that the data were obtained in anesthetized animals. The advantage of this approach was that differential responses of renal and adrenal sympathetic outputs could be recorded simultaneously, and therefore the comparisons were much more reliable than those recorded in different animals (32). Unfortunately, anesthesia decreases HR responses, especially the vagal component, and may qualitatively change the responses to microinjections of glutamate into the NTS. For example, glutamate microinjected into the NTS in conscious animals evokes increases in MAP and bradycardia, simulating chemoreflex responses (8-10), whereas in anesthetized animals it uniformly evokes decreases in MAP, HR, and sympathetic activity, simulating baroreflex responses (15, 17, 31-33, 40). This suggests that in conscious animals exogenous glutamate activates predominantly NTS chemoreflex mechanisms, whereas in anesthetized animals activation of baroreflex mechanisms prevail. Therefore, our data may reflect mostly the interactions between purinergic and glutamatergic neurotransmission/neuromodulation in NTS baroreflex mechanisms. As we showed previously (32), baroreflex regulation of regional sympathetic outputs is well preserved under the same conditions of chloralose-urethan anesthesia used in the present study, and these results are consistent with data obtained in conscious animals (32).

The results of the present study are limited to the interaction between glutamatergic transmission in the NTS and selective activation of two subtypes of purinergic receptors P2x and A2a via microinjections of their stable, exogenous agonists. These specific purinergic receptors were chosen because their stimulation affects NTS cardiovascular mechanisms in a baroreflex-like manner and because glutamate is a primary, fast neurotransmitter in the baroreflex arc. It is possible that synaptically released ATP and adenosine may interact differently with glutamatergic mechanisms. The natural action of ATP is very dynamic and complex. When released, it stimulates ligand gated channels (P2x) and may simultaneously evoke neuromodulatory responses via P2y receptors coupled to G proteins (11, 30). In addition, ATP is rapidly catabolized to adenosine, which can act via pre- or postsynaptic A1, A2a, and A3 receptors eliciting diverse neuromodulatory effects. Moreover, available antagonists of P2x and A2a receptors are either not very selective (such as suramin or pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid) (30) or require solvents that by themselves evoke marked hemodynamic responses on microinjection into the NTS (such as DMSO, a solvent for the A2a receptor antagonist CGS-15943) (4). Considering these complex and dynamic interactions between the natural agonists of P2x and A2a receptors and the lack of selective, water-soluble antagonists for these receptors, experiments with the respective antagonists for ATP and adenosine may give inconclusive results at this time. Our present data clearly indicate the possible interactions or lack of such an interaction between NTS P2x, A2a, and ionotropic glutamatergic mechanisms. However, assessment of naturally occurring interactions between these receptors on their synaptic activation awaits further investigation.

Unilateral blockade of NTS ionotropic glutamatergic transmission changed the resting levels of all recorded parameters in nonuniform ways (Table 2), slightly complicating comparisons between the responses obtained under control and blockade conditions. After the blockade, the slow components of hemodynamic and neural responses to P2x receptor stimulation had not been markedly changed compared with those under control conditions. This suggests that moderate, nonuniform shifts in baseline MAP, HR, and RSNA did not markedly affect the responses to P2x and A2a agonists. Although the marked increase in pre-ASNA after the blockade did not attenuate slow responses to P2x receptor stimulation, it may have limited further increases in pre-ASNA in response to A2a receptor stimulation. A small decrease in HR accompanied the unilateral blockade of NTS ionotropic receptors, which seems to be paradoxical with respect to baroreflex impairment. However, the blockade of ionotropic glutamatergic transmission at the level of the NTS or ventrolateral medulla, which results in impairment of baroreflex responses, does not change or even decreases HR in rats (15, 17). We previously reported (33) that severe impairment of HR baroreflex as a result of blockade of P2 receptors in the same site of the NTS was also accompanied by a small but statistically significant bradycardia. This bradycardia may be a result of simultaneous blockade of descending cardioacceleratory influences from higher centers that converge on NTS interneurons (38).

The effectiveness of ionotropic glutamatergic blockade was shorter than the slow responses to A2a receptor stimulation, and thus the analysis of these responses had to be limited to only 20 min, a time period during which the dose of KYN used in this study effectively impaired baroreflex responses on bilateral microinjection (Fig. 2). In addition, after only several minutes of relatively stable responses to KYN, all parameters started returning slowly toward control levels, and these effects were superimposed on the slow responses to P2x and A2a receptor stimulation. Supplementary doses of KYN could not be applied without interference with the slow responses to P2x and A2a receptor agonists. Therefore, the spontaneous changes in baseline parameters following unilateral ionotropic glutamatergic blockade were evaluated in a separate group of animals, and respective values were subtracted from the responses to P2x and A2a receptor stimulation as described earlier (Table 3). It should be stressed that the shifts in resting values did not affect the fast component of the response to P2x receptor stimulation. Despite the various effects of KYN on baseline hemodynamic and neural parameters (Table 2), the fast responses to alpha ,beta -MeATP were virtually abolished following the blockade.

In conclusion, blockade of ionotropic glutamatergic receptors in the subpostremal NTS virtually abolished the fast but not the slow component of the hemodynamic and differential neural responses evoked by stimulation of P2x receptors in the same site of the NTS. The blockade did not markedly attenuate the hemodynamic and maximal RSNA responses to stimulation of NTS A2a receptors; however, it likely limited further increase of pre-ASNA in this setting. These data suggest that the fast responses to stimulation of P2x receptors located in the subpostremal NTS may be mediated via facilitation of glutamatergic transmission. Slow, long-lasting responses to stimulation of P2x and A2a receptors are mostly independent of ionotropic glutamatergic transmission and may be mediated by the release of other slow-acting neuromodulators.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of C. Cupps. We also gratefully acknowledge the generous gift of arfonad by Hoffmann-La Roche, Nutley, NJ.


    FOOTNOTES

This study was supported by National Institutes of Health Grants MH-47181 and HL-02844.

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, School of Medicine, Wayne State Univ., 540 E. Canfield Ave., Detroit, MI 48201 (E-mail: doleary{at}med.wayne.edu).

Received 3 September 1999; accepted in final form 6 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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