Adenosine A2A receptors in the rostral ventrolateral medulla participate in blood pressure decrease with electroacupuncture in hypertensive rats

Figure 1. Timeline of experiments for four groups of rats. The Dahl salt-sensitive (DS) rats were randomly divided into normotensive control, EA, sham-EA, and untreated hypertension groups. All DS rats were restrained for 30 min twice weekly throughout the experiment. Normotensive control rats were fed a regular diet over the experimental period. In hypertensive groups, two weeks after restrained training, DS rats were given the 4% high salt diet in the three groups for nine weeks. After feeding with a 4% high salt diet for five weeks, the DS rats were treated with EA, sham-EA at the ST36-37 acupoints, or restraint only without the needle insertion for 30 min, twice weekly for another four weeks in one of these three groups, respectively. Blood pressure (BP) in all group rats was evaluated weekly using a tail-cuff device. Twenty-four hours after terminating the treatment, the rVLM microinjections were performed in rats treated with EA, sham-EA, or normal controls to examine a role of A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM)”. Besides, rats from each hypertensive group were decapitated to examine the expression of A2AR in the rVLM.

Figure 2. Blood pressure (BP) in Dahl salt-sensitive (DS) rats. Hypertension was developed similarly five weeks after a 4% high salt diet in all three hypertensive groups. Systolic BP (SBP) and diastolic BP (DBP) of DS rats remained elevated for another four weeks in the non-treatment and sham-EA groups. There was no difference in SBP and DBP between these two groups. In contrast, elevated SBP and DBP in the EA-treated group were significantly decreased after two weeks of EA treatment and remained reduced during the remaining course of EA therapy. SBP and DBP of DS rats fed with a regular diet were unaltered throughout the 11 weeks. Values represent means ± SE. The symbols +, &, and @ indicate P < 0.05, after vs. before onset of consumption of the high salt diet in hypertension control (o), sham-EA (●), and EA (*), respectively. # and $ correspondingly show P < 0.05, EA vs. hypertension control and sham-EA. BPs in three hypertensive groups were higher than in normotensive controls from week 5–week 11 (all P < 0.05).

Figure 3. Involvement of A2A receptors (A2AR) in the rVLM in EA modulation of high blood pressure (BP) in dahl salt-sensitive hypertensive rats (DSHRs). (A) and (B) changes in BP induced by the microinjection of DMSO (the vehicle for SCH 58261; Panel A) or SCH 58261 (an A2AR antagonist; 1 mM, 50 nl; Panel B) into the rVLM of EA-treated DSHRs. In (A,B), examples of original BP tracings before and after the microinjection are displayed above the bar graphs. Arrows below the tracing in (A,B) indicate the time of the microinjection into the rVLM.

Figure 4. Influence of activating A2A receptors (A2AR) in the rVLM on high blood pressure (BP) in dahl salt-sensitive hypertensive rats (DSHRs) treated with sham-EA. (A,B) Alterations in BP following the microinjection of DMSO (the vehicle for CGS-21680; Panel A) or CGS-21680 (an A2AR agonist; 0.4 mM, 50 nl; Panel B) into the rVLM of DSHRs treated with sham-EA. * P < 0.05, after vs. before the microinjection. In (A,B), examples of original BP tracings before and after the microinjection are displayed above bar graphs. Arrows below the tracing in (A,B) indicate the time of the microinjection into the rVLM.

Figure 5. Anatomic locations of microinjection sites in the rat. Top and middle panels: Composite maps displaying histologically verified sites of microinjections in the rVLM of Dahl salt-sensitive rats. The brain section shows the composite of planes of the medulla oblongata (Paxinos and Watson's atlas). Symbols represent the microinjection of DMSO (the vehicle, ●), SCH 58261 (⋆), CGS-21680 (□), and injections outside the rVLM (▪). All microinjections were performed unilaterally, with the sides chosen at random. rVLM, rostral ventrolateral medulla; Sp5, spinal trigeminal nucleus; Amb, nucleus ambiguus. Bottom panel: An original slide of the medulla oblongata (Bregma −12.12 mm) shows a track of a microdialysis probe insertion used for injections, indicated by two short arrows. A long arrow indicates the site of the microinjection in the rVLM.

Figure 6. Protein expression of A2A receptors in the rVLM of dahl salt-sensitive hypertensive rats. (A) Original bands display protein levels of A2A receptors (A2AR) in the rVLM from three rats, one from each hypertensive experimental group. (B) Group data show that the protein expression of rVLM A2AR 24 h after terminating EA was increased relative to controls and sham-EA. The relative value of A2AR/β-Actin is shown in the bar graph (n = 5 for each group). Symbols +, * indicate significant differences (P < 0.05) compared respectively with hypertensive controls and animals treated with sham-EA.