Figure 1. CO2/H+-induced vasoconstriction of RTN arterioles is mediated by a purinergic dependent mechanism involving P2Y2/4 receptors.
(A) trace of an RTN arteriole diameter show that increasing CO2 in the perfusion media from 5% to 15% (balance air, in TTX) caused vasoconstriction under baseline conditions but not in PPADS (5 µM). (B) example vessel image under baseline conditions and corresponding fluorescent intensity profile plots also show that exposure to high CO2 decreased vessel diameter. Profile plot scale bars: 2000 a.u., 10 µm. (C) summarized results of RTN arteriole responses to CO2/H+ under baseline conditions (N = 34 vessels) and when P2-receptors were blocked (5 µM PPADS; N = 8 vessels), P1-receptors were blocked (10 µM 8-PT; N = 7 vessels), or ectonucleotidase activity was inhibited (100 µM POM1; N = 5 vessels). (D) example diameter traces show RTN arterioles constrict in response to bath application of ATP (100 µM) or the selective P2Y2/4 receptor agonist UTPγS (0.5 µM) but dilate when P1 receptors are activated by adenosine (Ado; 1 µM). (E) summary data plotted as % diameter change in response to ATP (N = 7 vessels), UTP (N = 8 vessels), α,β-mATP (100 µM, preferential P2X agonist; N = 9 vessels) or adenosine (N = 9 vessels). (F–G), immunoreactivity for P2Y2 (F) and P2Y4 (G) receptors was detected as brightly label puncta near endothelial cells (DyLight 594 Isolectin B4 conjugate; IB4), arteriole smooth muscle (α-smooth muscle actin; αSMA), and astrocytes (glial fibrillary acidic protein; GFAP) associated with arterioles in the RTN (N = 3 animals). Arrows identify receptor labeling close to endothelial or smooth muscle cells and arrowhead identifies receptor labeling of astrocyte processes. Scale bar 10 µM. Hash marks designate a difference in µm from baseline as determined by RM-one-way ANOVA and Fishers LSD test or paired t-test and asterisks identify differences in CO2/H+-induced % change under baseline conditions vs in the presence of PPADS (C) or ATP vs specific agonist-induced % change (E) (one-way ANOVA and Fishers LSD test); one symbol = p<0.05, two symbols = p<0.01, three symbols = p<0.001, four symbols = p<0.0001.