NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism

Abstract

Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism.

Keywords: adrenal nerve; lumbar nerve; nucleus of the solitary tract; purinergic receptors; renal nerve; γ-aminobutyric acid.

Fig. 1.

Timeline of experiments. Small arrows indicate bolus injections of phenylbiguanide (PBG) in increasing doses (1–8 μg/kg) into the right atrium in ∼2.5-min intervals. Double large arrows represent bilateral microinjections into the nucleus tractus solitarii (NTS) of 1) artificial cerebrospinal fluid (ACF; 100 nl) or the GABA_A receptor antagonist bicuculline (Bic; 10 pmol/100 nl) or the GABA_B receptor antagonist SCH-50911 (SCH; 1 nmol/100 nl) followed by the adenosine A_2a receptor agonist CGS-21680 (CGS; 20 pmol in 50 nl) or 2) the GABA_A receptor antagonist alone [Bic (10 pmol/100 nl)] or the GABA_B receptor antagonist alone [SCH (1 nmol/100 nl)].

Fig. 2.

Microinjection sites for all experiments were located within the subpostremal NTS, as shown in schematic diagrams of transverse sections of the medulla oblongata from a rat brain. AP, area postrema; c, central canal; 10, dorsal motor nucleus of the vagus nerve; 12, nucleus of the hypoglossal nerve; Ts, tractus solitarius; Gr, gracile nucleus; Cu, cuneate nucleus. Scale is shown at the bottom; numbers on the left of the schematic diagram denote the rostrocaudal position in millimeters of the section relative to the obex according to the atlas of the rat subpostremal NTS by Barraco et al. (4). Bilateral microinjection sites for different pharmacological agents were marked with fluorescent dye. A: ■, ACF (100 nl) + CGS (20 pmol/50 nl); □, Bic (10 pmol/100 nl) + CGS (20 pmol/50 nl); and ▲, SCH (1 nmol/100 nl) + CGS (20 pmol/50 nl). B: ○, Bic (10 pmol/100 nl); and ●, SCH (1 nmol/100 nl).

Fig. 3.

Examples of cardiopulmonary chemoreflex (CCR) responses in mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve activity (RSNA), adrenal sympathetic nerve activity (ASNA), and lumbar sympathetic nerve activity (LSNA) evoked by intra-atrial injections of PBG (1–8 μg/kg) under control conditions (left) and after stimulation of NTS adenosine A_2a receptors preceded by volume control [CGS (20 pmol/50 nl) + ACF (100 nl); middle] or after stimulation of adenosine A_2a receptors preceded by GABA_A receptor blockade [Bic (10 pmol/100 nl) + CGS (20 pmol/50 nl); left]. Left and right: recordings preformed in one animal; middle: powerful inhibition of CCR responses after activation of A_2a receptors recorded in the other animal (ACF + CGS). GABA_A receptor blockade markedly attenuated A_2a receptor-mediated inhibition of CCR responses.

Fig. 4.

Comparisons of CCR responses obtained under control conditions (●) versus responses obtained after microinjections of either volume control [ACF (100 nl)] or GABA_A [Bic (10 pmol/100 nl)] or GABA_B [SCH (1 nmol/100 nl)] receptor antagonists followed by A_2a receptor agonist [CGS (20 pmol/50 nl); ○]. Data are means ± SE. *P < 0.05 vs. control; #P < 0.05, significant parallel shift of the experimental versus control curves without a significant PBG dose versus experimental condition interaction. Activation of A_2a receptors after volume control showed powerful inhibition of hemodynamic and regional sympathetic CCR responses. GABA_A receptor blockade attenuated adenosine A_2a receptor-mediated inhibition of CCR responses, whereas GABA_B receptors had a smaller effect.

Fig. 5.

Relative inhibition of hemodynamic and regional sympathetic reflex responses evoked by stimulation of NTS adenosine A_2a receptors [CGS (20 pmol/50 nl)] after pretreatment with volume control (ACF + CGS), GABA_A receptor blockade [microinjections of Bic (10 pmol/100 nl) (Bic + CGS)], or GABA_B receptor blockade [microinjections of SCH (1 nmol/100 nl) (SCH + CGS)] at four different levels of activation of the reflex [PBG (1–8 μg/kg)]. Data are means ± SE. SNA, sympathetic nerve activity (renal, adrenal, and lumbar). *P < 0.05, significant experimental effect versus ACF + CGS; #P < 0.05 vs. zero. GABA_A receptor blockade removed A_2a receptor-mediated inhibition of reflex responses to a much greater extent than GABA_B receptor blockade.

Fig. 6.

Comparisons of CCR responses obtained under control conditions (●) versus responses obtained after NTS GABA_A or GABA_B receptor blockades [microinjections of Bic (10 pmol/100 nl) or SCH (1 nmol/100 nl), respectively; ○]. Data are means ± SE. GABA_A receptor blockade tended to facilitate reflex responses (downward shifts of the reflex function curves obtained after the blockade).

Fig. 7.

Relative changes of hemodynamic and regional sympathetic reflex responses evoked by GABA_A or GABA_B receptor blockade in the NTS [microinjections of Bic (10 pmol/100 nl) or SCH (1 nmol/100 nl), respectively] at four different levels of activation of the CCR [intra-atrial PBG (1–8 μg/kg)]. Data are means ± SE. *P < 0.05, significant experimental effect vs. Bic; #P < 0.05 vs. zero. The negative deflections of the bars correspond to facilitation of the reflex compared with the respective control conditions. GABA_A receptor blockade facilitated reflex responses; the relative changes in reactivity were significantly different from zero, especially for moderate activation of the reflex.

Fig. 8.

Examples of the effect of bilateral microinjections of a high dose of Bic (100 pmol/100 nl) into the right (R) and left (L) sides of the NTS on baseline values of MAP, HR, ASNA, and LSNA recorded in one animal. Approximately 10 s after the microinjections, all baselines become markedly unstable.

Fig. 9.

Potential fine tuning of the CCR by adenosine released into the NTS. Th diagram shows direct and indirect adenosinergic inhibition of the CCR via A₁ (inhibitor) and A_2a (facilitator) receptors under conditions when large activation of the CCR is becoming detrimental for the organism and may lead to the release of adenosine into the NTS. Adenosine A₁ receptors directly inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A_2a receptors facilitate the release of GABA, which, in turn, inhibits transmission in the reflex pathway. Note that moderate activation of the CCR is cardioprotective and does not lead to the release of adenosine into the NTS.