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J Appl Physiol 103: 1205-1211, 2007. First published July 26, 2007; doi:10.1152/japplphysiol.01420.2006
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Activation of central adenosine A2A receptors enhances superior laryngeal nerve stimulation-induced apnea in piglets via a GABAergic pathway

Jalal M. Abu-Shaweesh

Department of Pediatrics, Case Medical Center and Case Western Reserve University, Cleveland, Ohio

Submitted 14 December 2006 ; accepted in final form 13 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Activation of the laryngeal mucosa results in apnea that is mediated through, and can be elicited via electrical stimulation of, the superior laryngeal nerve (SLN). This potent inhibitory reflex has been suggested to play a role in the pathogenesis of apnea of prematurity and sudden infant death syndrome, and it is attenuated by theophylline and blockade of GABAA receptors. However, the interaction between GABA and adenosine in the production of SLN stimulation-induced apnea has not been previously examined. We hypothesized that activation of adenosine A2A receptors will enhance apnea induced by SLN stimulation while subsequent blockade of GABAA receptors will reverse the effect of A2A receptor activation. The phrenic nerve responses to increasing levels of SLN stimulation were measured before and after sequential intracisternal administration of the adenosine A2A receptor agonist CGS (n = 10) and GABAA receptor blocker bicuculline (n = 7) in ventilated, vagotomized, decerebrate, and paralyzed newborn piglets. Increasing levels of SLN stimulation caused progressive inhibition of phrenic activity and lead to apnea during higher levels of stimulation. CGS caused inhibition of baseline phrenic activity, hypotension, and enhancement of apnea induced by SLN stimulation. Subsequent bicuculline administration reversed the effects of CGS and prevented the production of apnea compared with control at higher SLN stimulation levels. We conclude that activation of adenosine A2A receptors enhances SLN stimulation-induced apnea probably via a GABAergic pathway. We speculate that SLN stimulation causes endogenous release of adenosine that activates A2A receptors on GABAergic neurons, resulting in the release of GABA at inspiratory neurons and subsequent respiratory inhibition.

control of breathing; sudden infant death syndrome hypotension

ACTIVATION OF THE LARYNGEAL mucosa either chemically (through instillation of water or acid) or mechanically results in apnea that is usually associated with adduction of upper airways and swallowing movements. This potent inhibitory reflex apnea, traditionally termed the laryngeal chemoreflex, has been well characterized in newborn infants (24, 25) and animals of different species and is mediated through the superior laryngeal nerve (SLN), a branch of the vagus nerve. Electrical stimulation of the SLN can induce a similar apneic response. The laryngeal chemoreflex is developmentally regulated because it is strongest in newborns (21) and has been proposed to play a role in the pathogenesis of apnea of prematurity (25), gastroesophageal reflux-induced apnea (17) and sudden infant death syndrome (6, 33). However, the exact mechanisms, neuronal connections, and neurotransmitters involved in this response have not been well characterized.

The maturation of reflex apnea in piglets is very well described, and newborn animals are known to exhibit an exaggerated response to laryngeal stimulation (18). We have established in our previous studies the involvement of {gamma}-aminobutyric acid (GABA) in SLN stimulation-induced apnea. Blockade of GABAA receptors through intracisternal or intravenous injections of bicuculline resulted in attenuation of SLN stimulation-induced apnea in newborn piglets (1). Theophylline has also been shown to prevent the laryngeal chemoreflex in newborn piglets (18). The methylxanthines caffeine and theophylline are widely used in the treatment of apnea of prematurity and have been traditionally thought to exert their action centrally by blocking receptors of the inhibitory neuromodulator adenosine (7, 12). Recent reports have documented an interaction between adenosine and GABA during regulation of breathing and sleep. We have reported that blockade of GABAA receptors abolishes the inhibitory effect of the adenosine A2A agonist CGS-1680 (CGS) on phrenic activity in piglets (34). Additionally, A2A receptors were found to colocalize with GABAergic neurons in the medulla oblongata of both piglets (34) and rats (35). Furthermore, activation of adenosine A2A receptors caused increased release of GABA in the tuberomammillary nucleus of rats (13). These data together with the ability of both bicuculline and theophylline to block SLN stimulation-induced apnea raise the possibility of an interaction between GABA and adenosine in the pathogenesis of this inhibitory reflex response, although such interaction has not yet been described.

We hypothesized that 1) activation of central adenosine A2A receptors will enhance the inhibition of phrenic activity induced by SLN stimulation and allow apnea to occur at lower levels of electrical stimulation and 2) subsequent blockade of GABAA receptors will reverse the effects of activation of adenosine A2A receptors. In this study, we aimed to test this hypothesis by 1) examining the effect of intracisternal administration of the adenosine A2A receptor agonist CGS on SLN stimulation-induced apnea and 2) evaluating the effect of subsequently administering the GABAA receptor antagonist bicuculline on this response in the newborn piglet.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine. The experiments were performed in 3- to 7-day-old piglets (n = 10), a well-identified model for studying laryngeal reflex apnea. The average weight of the animals was 2.1 ± 0.1 kg. The animals were initially sedated with an intramuscular mixture of xylazine (2.8 mg/kg) and ketamine (14.4 mg/kg), followed by intravenous thiopental sodium at a dose of 25–30 mg/kg. The trachea was intubated through a cervical tracheostomy and the animals were placed on a volume-controlled ventilator (Harvard Apparatus). The volume and frequency of tidal breaths were adjusted to keep a stable eucapnic arterial PCO2 (PaCO2; 35–45 Torr), using an end-tidal CO2 analyzer, while using an inspired O2 concentration of 100%. Femoral venous and arterial lines were inserted for administration of fluid and medication, and for blood pressure monitoring and blood gas analysis, respectively.

To avoid the known confounding effects of anesthesia on breathing and laryngeal reflex apnea (8, 18, 21), midcollicular decerebration was performed as previously described in newborn piglets (1, 5). Anesthesia was discontinued after decerebration was completed, and the animals were allowed to recover for at least 1 h following the surgical preparation before any experimental recordings were performed. Gallamine triethiodide (10 mg·kg–1·h–1) was used for paralysis.

Both vagal nerves were identified and cut in the caudal cervical area, below the origin of the SLNs to avoid the effect of lung inflation and other afferent pulmonary inputs on respiratory timing. The phrenic nerve was dissected, sectioned, desheathed, and placed on a bipolar recording electrode. The phrenic nerve electroneurogram was amplified (model P511, Grass Instruments) and rectified, and the moving average signal was connected to an analog computer. The SLN on one side was identified, dissected, sectioned, desheathed, and placed on a bipolar electrode connected to a stimulator (model S11, Grass Instruments). We defined SLN threshold, as the minimal amount of electrical current needed to decrease phrenic nerve amplitude or frequency. The electrical current needed to induce threshold stimulation averaged 6.6 ± 0.6 with a range of 4–11 µA. Fifteen seconds of SLN stimulation was performed at threshold, 1.5, 2, and 4x threshold using impulses with pulse duration of 4.5 ms and pulse interval of 50 ms applied through a photoelectric isolation unit (model PSIU6, Grass Instruments).

The animals were placed prone, the back of the neck was dissected, and muscles were separated in the midline. The occipitoatlantic membrane was visualized, and the tip of a 10-to 15-cm, 25-gauge soft venous catheter was inserted through the membrane. Proper placement was confirmed by cerebrospinal fluid leak. The phrenic nerve response to multiple levels of SLN stimulation was measured at baseline and after sequential intracisternal administration of 1) 0.1–0.3 ml of DMSO, the vehicle for CGS (n = 6); 2) 0.1–0.3 ml of a 10 µM solution of adenosine A2A receptor agonist CGS (n = 10); and 3) 0.1–0.2 ml of 1 mg/ml solution of the GABAA receptor blocker bicuculline (n = 7). These concentrations were chosen based on the minimal dose needed to cause a consistent effect from our previous studies in piglets (1, 34). The average duration between CGS and bicuculline administration was 118 ± 18 min. This represented the duration needed for adjustment of ventilator rate to allow for recovery of phrenic activity following CGS administration plus the duration of the SLN stimulation.

Data Collection and Statistical Analysis

We used PowerLab hardware and Chart software (both from AD Instruments, Mountain View, CA) to capture signals in real time (2-kHz sampling rate). Blood pressure and full-wave rectified and moving averaged phrenic neurogram were recorded, as well as airway CO2 during both inspiration and expiration. The integrated moving average of phrenic nerve activity was analyzed for phrenic area, frequency, inspiratory (TI) and expiratory times (TE), and minute activity (phrenic area times frequency). All phrenic parameters were averaged for 1 min during baseline (before any intervention), 30 s preceding each SLN stimulation, and the 15-s duration of each SLN stimulation at threshold and at 1.5, 2, and 4x threshold. Data are presented as means ± SE of normalized values relative to prestimulation levels (average value during 15 s of stimulation/average value during 30 s preceding stimulation x 100%). The effect of the administered chemical on the phrenic activity was also normalized to values before administration using the above formula. Apnea was defined as doubling of TE compared with baseline.

Repeated-measures two-way ANOVA was used to compare the effect of CGS and bicuculline on the phrenic response to SLN with post hoc comparisons using Student-Neuman-Keuls test. One-way ANOVA was used to evaluate the effect of different interventions on phrenic minute activity, frequency, tidal volume, as well as mean arterial blood pressure, heart rate, and PaCO2 levels after different interventions. Pearson's coefficient (P) value of <0.05 was used for statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
SLN Stimulation

As we have previously described, SLN stimulation caused a decrease in phrenic area, frequency, and minute phrenic activity that was proportional to the level of stimulation. The decrease in phrenic frequency was secondary to a significant prolongation of the TE while there was a significant shortening of TI with increasing stimulation secondary to the occurrence of apnea (Table 1). The incidence and duration of apnea in response to SLN stimulation also increased in response to higher levels of stimulation (Table 1).


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  		Table 1. Effect of increasing levels of SLN stimulation on phrenic activity

 
DMSO (CGS vehicle).   In the first six animals, the phrenic responses to graded SLN stimulation were measured following intracisternal administration of DMSO, the vehicle for CGS. Phrenic nerve responses to SLN stimulation following DMSO were identical to those of the control period. Therefore, in the last four animals, this step was omitted. The responses to CGS and bicuculline administration in all animals presented hereafter were compared with the control levels.

Activation of Central A2A Receptors

The effect on baseline phrenic activity.   Intracisternal CGS administration caused a significant decrease in phrenic nerve activity and resulted in apnea in four animals, (Fig. 1). In seven animals, recovery of phrenic activity required an increase in PaCO2 that was achieved by decreasing the ventilator rate. This resulted in an increase in average PaCO2 in all the animals from 39.7 ± 1.4 to 46.4 ± 3.3 Torr before and after CGS, respectively (P = 0.017, 1-way ANOVA) and in recovery in phrenic activity such that phrenic area and frequency were 66 ± 12% and 121 ± 10% of pre-CGS levels, respectively [not significant (NS)].


Figure 1
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  		Fig. 1. Effect of CGS on baseline phrenic activity and blood pressure (BP). Under similar end-tidal CO2 (ETCO; in Torr) levels, CGS decreased both systolic and diastolic BP and phrenic activity. After ETCO2 was increased (not shown) by decreasing the ventilator rate, only the phrenic activity recovered, whereas the decrease in BP persisted.

 
The effect on blood pressure and heart rate.   Intracisternal CGS administration caused a significant decrease in mean arterial blood pressure (MAP) secondary to a decrease in both systolic and diastolic pressures (Fig. 1). The MAP was 54.7 ± 2.1 and 40.3 ± 4 mmHg before and after CGS administration (P < 0.01, 1-way ANOVA). Furthermore, CGS caused a significant increase in heart rate from 193 ± 11 to 273 ± 8 beats/min (P < 0.001, 1-way ANOVA).

The effect of CGS on the laryngeal reflex.   As during control stimulation, increasing levels of SLN stimulation caused a significant decrease in phrenic area, frequency, minute phrenic activity, and TI, as well as a significant prolongation of TE and duration of apnea (all P < 0.01). Additionally, compared with control, CGS administration caused significantly more inhibition of phrenic activity in response to increasing levels of SLN stimulation (Fig. 2). CGS administration exaggerated both the decrease in phrenic area, frequency, minute phrenic activity, and TI and the prolongation of TE and duration of apnea responses to increasing levels of SLN stimulation with a significant interaction between the effect of CGS administration and SLN stimulation (P < 0.01 for all, 2-way ANOVA; Figs. 3 and 4). The effect of CGS administration was more obvious at lower levels of stimulation threshold (1.5x threshold than at 2x and 4x threshold), probably secondary to the higher stimulations causing apnea in the majority of animals during the control period (Figs. 3 and 4). However, the duration of apnea following CGS administration was significantly longer compared with control in response to all levels of SLN stimulation beyond threshold (P < 0.001 for all paired comparisons; Fig. 5). The number of animals exhibiting apnea at each level of stimulation was also significantly higher than during control (P < 0.05; Fig. 5).


Figure 2
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  		Fig. 2. Effect of CGS on the phrenic nerve responses to superior laryngeal nerve (SLN) stimulation. CGS administration enhanced the inhibitory effects of SLN stimulation on phrenic activity. A: at 1.5x threshold, 15-s stimulation of the SLN caused a mild decrease in phrenic activity under control conditions and apnea after CGS. B: at 2x threshold, 15-s stimulation of the SLN caused apnea under control conditions that lasted longer and extended beyond the duration of stimulation after CGS.

 

Figure 3
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  		Fig. 3. The effect of CGS and bicuculline on the phrenic area and frequency responses to SLN stimulation. Increasing levels of SLN stimulation caused progressive inhibition of phrenic area and frequency that was significantly more pronounced after CGS administration. Bicuculline reversed the enhanced decreased in phrenic area and frequency in response to SLN stimulation seen after CGS. Furthermore, after bicuculline administration, 2x and 4x threshold stimulation caused a smaller decrease in the phrenic area and frequency, respectively compared with control (P < 0.05). The effect of different levels of stimulation depended on CGS and bicuculline administration with a statistically significant interaction between SLN stimulation and the administration of CGS vs. both control and bicuculline (P < 0.001) but not between SLN stimulation and bicuculline administration vs. control (2-way ANOVA). Post hoc analysis of the effect at different levels of stimulation: *P < 0.05 vs. CGS the at same stimulation level; {ddagger}P < 0.05 vs. control at same stimulation level.

 

Figure 4
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  		Fig. 4. The effect of CGS and bicuculline on phrenic respiratory timing responses to SLN stimulation. There was no effect of either CGS or bicuculline on baseline respiratory times. CGS administration enhanced the prolongation of expiratory time and shortening of inspiratory time in response to SLN stimulation while bicuculline reversed the effects of CGS on respiratory timing. Furthermore, bicuculline prevented the prolongation of expiratory time and shortening of inspiratory time in response to 4x threshold stimulation compared with control. The effect of different levels of stimulation depended on CGS and bicuculline administration with a statistically significant interaction between SLN stimulation and CGS administration vs. control and bicuculline (P < 0.01) but not between SLN stimulation and bicuculline administration vs. control (2-way ANOVA). Post hoc analysis of the effect at different levels of stimulation: *P < 0.05 vs. CGS the at same stimulation level; {ddagger}P < 0.05 vs. control at same stimulation level.

 

Figure 5
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  		Fig. 5. The effect of CGS and bicuculline on the incidence and duration of SLN stimulation induced apnea. A: CGS administration caused prolongation of the duration of apnea following SLN stimulation, whereas bicuculline inhibited the production of apnea except at higher levels of stimulation. The effect of different levels of stimulation depended on CGS and bicuculline administration with a statistically significant interaction between SLN stimulation and all 3 interventions (P < 0.01). Post hoc analysis of the effect at different levels of stimulation: *P < 0.05 vs. CGS the at same stimulation level; {ddagger}P < 0.05 vs. control at same stimulation level. B: CGS increased the number of animals with apnea at all levels of stimulation compared with control. Bicuculline completely prevented SLN-induced apnea except at higher levels of stimulation and in fewer animals compared with CGS. *P < 0.05 vs. control and bicuculline.

 
Subsequent Blockade of GABAA Receptors

The effect on baseline phrenic activity.   Intracisternal administration of bicuculline following CGS caused recovery of phrenic activity that allowed for a decrease of PaCO2 from the high levels needed after CGS administration toward control values. PaCO2 following bicuculline was 40 ± 2 Torr (P < 0.05 and 0.66 vs. CGS and control, respectively). Even after PaCO2 was lowered to control levels, bicuculline still caused a significant increase in phrenic area to 192 ± 50% (P < 0.05), compared with prebicuculline, post-CGS levels, whereas phrenic frequency and minute phrenic activity did not change (79 ± 10% and 135 ± 21%, respectively; NS). However, phrenic area, frequency, and minute activity following bicuculline were not different from control, pre-CGS levels; i.e., bicuculline blocked the effects of CGS and returned phrenic activity toward control values. Following bicuculline administration, the phrenic area, frequency, and minute activity were 83 ± 14, 89 ± 8, and 77 ± 16% respectively compared with control pre-CGS levels (NS).

The effect on blood pressure and heart rate.   Intracisternal bicuculline caused recovery of MAP toward control, pre-CGS levels. The MAP was 50 ± 5.9 mmHg following bicuculline (P < 0.01 and 0.71 vs. CGS and control, respectively). However, the tachycardia seen after CGS continued following bicuculline administration despite recovery of blood pressure. The heart rate was 287 ± 6 beats/min following bicuculline, significantly higher than control (P < 0.001) but not different from CGS (P = 0.13).

The effect on the laryngeal reflex.   The overall effect of bicuculline was to reverse the enhanced inhibition induced by SLN stimulation after CGS toward control pre-CGS values. Although SLN stimulation continued to cause significant decrease in phrenic area and minute ventilation after bicuculline, these responses were significantly less inhibited than after CGS administration (P < 0.001 for the interaction between the effects of bicuculline administration and SLN stimulation vs. CGS, 2-way ANOVA). Additionally, after bicuculline administration SLN stimulation failed to produce a significant effect on phrenic frequency, TI, or TE (Figs. 3 and 4). Furthermore, bicuculline prevented the inhibitory responses of the phrenic area and frequency to 2x and 4x threshold stimulation of the SLN, respectively, compared with both control and CGS responses (Figs. 3 and 4). Phrenic frequency and minute ventilation were 59 ± 20% and 32 ± 15% of baseline, respectively, following 4x threshold stimulation, whereas phrenic area was 58 ± 16% of baseline following 2x threshold stimulation (P < 0.05 for all vs. both control and CGS at the same stimulation level). The preservation of the frequency response to SLN stimulation was secondary to the ability of bicuculline to prevent prolongation of TE (Fig. 4). Bicuculline also decreased the duration of apnea in response to SLN stimulation compared with both control and CGS (P < 0.05 for the interaction between SLN stimulation and bicuculline administration vs. both control and CGS; Fig. 5). The duration of apnea at 4x threshold was 8.3 ± 6.2 s (P < 0.01 vs. CGS and control; Fig. 5). Furthermore, bicuculline caused apnea in fewer animals in response to all levels of SLN stimulation compared with CGS (P < 0.05 for all paired comparisons; Fig. 5).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The main findings of our study in the newborn piglet are that activation of central adenosine A2A receptors through intracisternal administration of CGS caused 1) inhibition of phrenic activity that recovered after PaCO2 was increased, 2) facilitation of the inhibitory effects of SLN stimulation on phrenic activity and apnea duration, and 3) hypotension and tachycardia. Subsequent blockade of GABAA receptors reversed the inhibitory effects of CGS on baseline phrenic activity, SLN stimulation induced depression of phrenic activity, and hypotension but not tachycardia. We have thus shown that activation of A2A receptors enhances SLN stimulation-induced apnea probably via a GABAergic pathway.

Activation of adenosine A2A receptors with CGS exaggerated the inhibitory effects of SLN stimulation on phrenic responses despite the hypercapnia that was needed to recover phrenic activity following CGS administration. This is of particular interest because hypercapnia increases the threshold for the production of the laryngeal chemoreflex (16, 19); i.e., the same level of SLN stimulation causes less inhibition of phrenic activity in the presence of hypercapnia. Another confounding variable that might have affected our results is the decrease in blood pressure following CGS administration. Although hypotension is part of the laryngeal chemoreflex in the intact animal, we are not familiar with any reports that link hypotension with an exaggeration of the laryngeal chemoreflex in the absence of metabolic derangements or respiratory inhibition. In our studies the decrease in blood pressure was not associated with metabolic acidosis, suggesting that there was no change in tissue perfusion. Additionally, because hypercapnia reversed the inhibition of the phrenic nerve and restored its activity to baseline levels, the exaggeration of SLN stimulation-induced apnea following CGS administration cannot be attributed to a nonspecific inhibition caused by CGS but rather to its ability to activate A2A receptors.

Bicuculline reversed the effects of CGS on phrenic nerve response to SLN stimulation and produced a reflex response that was less inhibited compared with control, pre-CGS level. The ability of bicuculline to attenuate SLN stimulation-induced apnea in the absence of a significant increase in phrenic activity and/or CO2 levels suggests that the bicuculline response was not secondary to a nonspecific excitation but rather to its ability to block GABAA receptors. Although bicuculline was administered almost 2 h after CGS, there was continuing evidence of CGS effect in the form of persistent phrenic inhibition, hypotension, and exaggerated phrenic depression in response to SLN stimulation just before administration of bicuculline. Therefore, the recovery of the phrenic activity and blood pressure following bicuculline administration was probably secondary to its ability to block the downstream effects of activation of A2A receptors by CGS. Furthermore, although bicuculline blocked the phrenic inhibition produced by higher levels of SLN stimulation compared with control, most of its effect was to reverse the exaggerated responses seen after CGS, signifying that bicuculline blocked the added effects of activation of adenosine A2A receptors. The residual inhibition of the phrenic activity in response to higher levels of SLN stimulation might suggest the presence of GABA-independent mechanisms or the need for higher dose of bicuculline in the presence of CGS as a similar dose of bicuculline was able to block this response in the absence of CGS (1).

Adenosine has been implicated in the regulation of breathing and is known to depress neural function (7). It is ubiquitously released (by Na+-dependent transporters) and is formed in the extracellular space by breakdown of released ATP (e.g., as a cotransmitter) and sequentially cleaved by ecto-ATPases/apyrases and ectonucleotidases. Four adenosine receptor subtypes (A1, A2A, A2B, and A3) have been cloned and pharmacologically characterized. Activation of A1 and A3 receptors decreases while activation of A2A and A2B receptors increases adenylyl cyclase activity, respectively (14). Analogs of adenosine have been shown to decrease respiration in neonatal rabbits and pigs (4, 10, 26). Furthermore, both A1 and A2A receptor agonists were found to mediate inhibition of fetal respiratory drive (12, 15, 32). Our data in newborn piglets support these observations and further implicate activation of adenosine A2A receptors in the production of apnea induced by SLN stimulation.

The methylxanthines theophylline and caffeine are widely used in neonatology practice to prevent and treat apnea of prematurity. Xanthines are thought to exert their action by blocking adenosine receptors, especially A1 and A2A, although the exact mechanism and location of action has not been identified. The involvement of adenosine in the laryngeal chemoreflex was initially suggested when theophylline was shown to block the reflex in newborn piglets (18). Our data suggest that methylxanthines are acting centrally by blocking adenosine A2A receptors on GABAergic neurons, thereby preventing the release of GABA and diminishing the respiratory inhibition induced by SLN stimulation; however, a role for adenosine A1 receptors cannot be excluded. We speculate that blockade of this pathway may contribute to the beneficial effects of xanthines in the treatment of apnea of prematurity.

GABA is the major inhibitory neurotransmitter in the central nervous system. It was found to inhibit breathing mainly via activation of GABAA receptors (9, 11). We have shown previously in newborn piglets that blocking GABAA receptors almost abolished apnea induced by SLN stimulation (1). There is an increasing body of evidence of a central interaction between adenosine and GABA. We have previously reported that blockade of GABAA receptors abolishes the inhibitory effect of the adenosine A2A agonist CGS on phrenic activity (34). The adenosine A2A receptor system appears to be one of the presynaptic neuromodulatory systems able to enhance the evoked release of GABA from hippocampal nerve terminals (3) and tuberomammillar nucleus in rats (13). Furthermore, anatomic studies have shown that A2A receptors colocalize with GABAergic neurons in regions of the medulla oblongata in piglets (34) and developing rats (35). These data as well as the ability of both theophylline and bicuculline to prevent the laryngeal reflex apnea point to the possible interaction between GABA and adenosine in the production of apnea during SLN stimulation. Our findings further expand these observations and allow for the speculation that SLN stimulation causes endogenous release of adenosine that activates A2A receptors on GABAergic neurons, resulting in the release of GABA at inspiratory neurons and subsequent respiratory inhibition.

Similar interaction between A2A and GABAA receptors can be proposed to explain the effects of CGS and bicuculline on the blood pressure. Activation of adenosine A2A receptors in the nucleus tractus solitarii as well as GABAA receptors in the rostral ventrolateral medulla (RVLM) have both been shown to cause hypotension and bradycardia through inhibition of sympathetic activity (20, 27–29). Furthermore, blockade of GABAA receptors in the sympathoexcitatory region of the RVLM resulted in an increase in sympathetic activity and blood pressure (2, 27, 31). These studies suggest a common pathway for hypotension induced by activation of GABAA and A2A receptors through inhibition of sympathetic activity, which is in agreement with our findings in vagotomized piglets. The ability of both CGS and bicuculline to regulate blood pressure despite vagotomy excludes a role for the parasympathetic, and it implicates the sympathetic system in this regulation. Unlike previously mentioned studies in which CGS caused bradycardia in anesthetized animals, intracisternal CGS caused tachycardia in our animals. This could be related to vagotomy or to lack of the confounding effect of anesthesia on the heart rate response to CGS, as was previously suggested. We speculate that SLN afferents activated during the laryngeal chemoreflex stimulate second-order neurons in the NTS that release adenosine, which in turn activates A2A receptors on GABA containing neurons resulting in the release of GABA at inspiratory and sympathetic regions causing apnea and hypotension. Our results do not exclude a role for other neurotransmitters and neuromodulators or other adenosine receptors in the laryngeal chemoreflex. Multiple other agents have been implicated in the genesis of apnea during laryngeal stimulation including glycine, beta-endorphins, and catecholamines (21, 22, 30). Furthermore, our findings do not directly indicate the involvement of endogenously released adenosine in the regulation of the laryngeal chemoreflex. However, the involvement of endogenous adenosine is suggested by the previously reported ability of theophylline to block the laryngeal chemoreflex.

Our study is further limited by its inability to identify the site of action of either CGS or bicuculline. However, the midcollicular decerebration performed in our studies together with the intracisternal administration of the chemicals through a catheter placed close to the surface of the medulla oblongata isolates the effect of CGS and bicuculline to the brain stem and especially the medulla. The variable responses obtained in different studies might be secondary to variable diffusion of the chemicals or to a dilution effect from the CSF. Further studies are clearly needed to identify the proposed network and document the interaction between adenosine and GABA in the production of SLN stimulation induced apnea.

In summary, we report in nonanesthetized newborn piglets that 1) central activation of A2A receptors caused an inhibition of phrenic activity, a decrease in blood pressure and tachycardia, and exaggeration of apnea induced by SLN stimulation despite hypercapnia and 2) subsequent central blockade of GABAA receptors reversed the effects of activation of A2A receptors on phrenic activity, SLN stimulation-induced apnea, and blood pressure. We conclude that both adenosine and GABA play a role in the respiratory and cardiovascular responses during laryngeal stimulation. We propose a pathway whereby this interaction takes place in the medulla.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants 5K08 HL-73240 and HL-62527.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
I thank Drs. Richard Martin and Musa Haxhiu for a stimulating discussion during the preparation of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Abu-Shaweesh, Div. of Neonatology, Dept. of Pediatrics, Rainbow Babies and Children's Hospital, 11100 Euclid Ave., Cleveland, OH 44106 (e-mail: JXA16{at}case.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
 ACKNOWLEDGMENTS
 REFERENCES
 

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