Adenosine Receptor A2a, but Not A1 in the rVLM Participates Along With Opioids in Acupuncture-Mediated Inhibition of Excitatory Cardiovascular Reflexes

FIGURE 1. Influence of blockade of adenosine receptors in the rVLM on EA modulation of GD-induced pressor reflexes. The diagram above bar graphs displays the sites of the P5 and P6 acupoints (Hua, 1994). Bars represent increases in mean arterial blood pressure (MAP) following GD. Values below each bar indicate the baseline MAP (means ± SE) before GD. (A,B) 5% DMSO (50 nl) was injected into the rVLM as sham-EA (A) or EA (B) was conducted at P5-6; (C) microinjection of 8-SPT (10 mM in 50 nl; a non-selective adenosine receptor antagonist) into the rVLM with EA treatment. ∗P < 0.05, a decrease of GD response after the onset of EA; #P < 0.05, after vs. before microinjection into the rVLM. Labels a-c in (C) indicate examples of the original BP tracings of a rat; ↓, time of GD application. These data suggest that adenosine in the rVLM is involved in EA modulation of GD-induced pressor reflexes.

FIGURE 2. Influence of specific blockade of A1 or A2a receptors in the rVLM on EA modulation of GD-induced pressor reflexes. Bars represent increases in mean arterial blood pressure (MAP) following GD. Values below each bar indicate the baseline MAP (means ± SE) before GD. (A,B) Microinjection of DPCPX (3 mM in 50 nl; an A1 receptor antagonist; A) or SCH 58261 (1 mM in 50 nl; an A2a receptor antagonist; B) into the rVLM with EA treatment. ∗P < 0.05, a decrease of GD response after the onset of EA; #P < 0.05, after vs. before microinjection into the rVLM. Labels a–d in (A) and e–h in (B) indicate examples of original BP tracings of a rat from each group; ↓, time of GD application. These data suggest that adenosine A2a, but not A1 receptors in the rVLM contribute to EA modulation of GD-induced pressor reflexes.

FIGURE 3. Influence of activation of A2a receptors in the rVLM on GD-induced pressor reflexes. Bars represent increases in mean arterial blood pressure (MAP) following GD. Values below each bar indicate the baseline MAP (means ± SE) before GD. (A–C) Microinjection of 5% DMSO (50 nl; A), SCH 58261 (1 mM in 50 nl; an A2a receptor antagonist; B) or CGS-21680 (0.4 mM in 50 nl; an A2a receptor agonist; C) into the rVLM without EA treatment. ∗P < 0.05, after vs. before microinjection into the rVLM. Labels a–c in (C) indicate examples of the original BP tracings of a rat from this group; ↓, time of GD application. These data suggest that the activation of adenosine A2a receptors in the rVLM contributes to inhibition of GD-induced pressor reflexes.

FIGURE 4. Importance of opioids in adenosine-mediated EA inhibition of GD-induced pressor reflexes through A2a receptors in the rVLM. Bars represent increases in mean arterial blood pressure (MAP) following GD. Values below each bar indicate the baseline MAP (means ± SE) before GD. (A) Sequential microinjections of normal saline and 5% DMSO (both 50 nl) into the rVLM, which are vehicles for naloxone (a non-selective opioid receptor antagonist) and SCH 58261 & CGS-21680 (an A2a receptor antagonist and agonist, correspondingly), respectively; (B–D) following microinjection of naloxone (1 mM in 50 nl) into the rVLM with EA treatment, 5% DMSO (the vehicle; B), SCH 58261 (1 mM in 50 nl; C) or CGS-21680 (0.4 mM in 50 nl; D) were administered into the same site of the rVLM. ∗P < 0.05, a decrease of GD response after the onset of EA; #P < 0.05, after vs. before microinjection of naloxone into the rVLM. Labels a–f in (C) indicate examples of the original BP tracings of a rat from this group; ↓, time of GD application. Blockade or activation of A2a receptors did not alter EA’s action in attenuating GD-evoked sympathoexcitatory cardiovascular reflexes after inhibition of opioid receptors in the rVLM.…

FIGURE 5. Anatomic locations of microinjection sites in the rat. (Top two panels) Composite maps displaying histologically verified sites of microinjections in the rVLM of rats. The box indicates the magnified area of the rVLM in these panels. Brain section shows the composite of planes of brain stem rostral to the obex (Paxinos and Watson’s atlas) (Paxinos and Watson, 2005). Symbols represent microinjection of 5% DMSO with and without normal saline (■), 8-SPT (⋄), DPCPX (+), SCH 58261 only (▲), CGS-21680 only (🌑), and naloxone (★). All the injections were unilateral (side chosen randomly). Sections are 1.08 – 1.32 and 1.32 – 1.56 mm rostral to the obex. Amb, nucleus ambiguous; IO, inferior olive; rVLM, rostral ventrolateral medulla; Sp5, spinal trigeminal nucleus. (Bottom panel) An original slide of the medulla oblongata (1.32 mm rostral to the obex) shows the blue-dyed track of a modified microdialysis probe insertion used for injection. Ventral aspect of blue dye represents the site of microinjection in the rVLM as indicated by an arrow. A scale bar represents 1 mm.

FIGURE 6. Confocal microscopic images show double-fluorescent labeling of adenosine A2a receptors and enkephalin in the rVLM of a rat. (A) Low-power photomicrograph (1.44 mm rostral to the obex). (B) Magnified region displayed within the box in (A). (B) Is a merged image from (C,D). Arrows in (B–D) indicate a neuron co-labeled with adenosine A2a receptors and enkephalin (yellow), adenosine A2a receptors (green) and enkephalin (red), respectively. Scale bars in (A,B–D) represent 200 and 50 μm, correspondingly.

FIGURE 7. Potential neural pathways associated with the action of adenosine A2a receptor-opioids in the rVLM in EA regulation of sympathetic outflow and cardiovascular pressor responses during visceral stimulation. +, indicates activation or enhancement of the system; –, indicates inhibition of the system; EA, electroacupuncture; GD, gastric distension. EA likely activates adenosine A2a receptors in the rVLM, which promotes opioid generation. The activation of opioid receptors causes inhibition of the pre-sympathetic activity of rVLM neurons directly as well as through suppression of A2a receptor-mediated excitation of these neurons, leading to a reduction in blood pressure.