P2Y2 receptors contribute to specialized CO2/H+‐dependent regulation of arteriole tone in the retrotrapezoid nucleus
Authors: Cleary Colin , Moreira Thiago , Mulkey Daniel 
Published: June 2021

Regulation of cerebral blood flow is critical for normal brain function; the accumulation of CO2/H+causes vasodilation leading to enhanced delivery of O2 and removal of CO2/H+, thus helping to match blood flow with tissue metabolic needs. Tissue CO2/H+ also functions as the primary stimulus for breathing by activating respiratory chemoreceptors. Interestingly, recent evidence suggests that CO2/H+‐dependent regulation of blood flow in a chemoreceptor region, known as the retrotrapezoid nucleus (RTN), is opposite to the rest of the cerebrovascular tree; exposure to high CO2/H+ decreases arteriole tone by a mechanism that may involve P2Y2 receptors. However, it is not clear which cells at the astrocyte‐arteriole interface express P2Y2 receptors. Making a determination of cell type and P2Y2 function is important regulation of blood flow in the RTN influences respiratory behavior. Therefore, the goals of this study are to confirm in vitro and in vivo that P2Y2 receptors are important determinants of RTN vascular tone, characterize P2Y2 mRNA expression within each component of the astrocyte‐arteriole interface in chemoreceptor regions including the caudal nucleus tractus solitarius (cNTS), raphe obscurus (ROb), and RTN by qRT‐PCR as well as characterizing P2Y2 dependent calcium signaling in vasculature in each brainstem respiratory area. Brainstem slices were isolated from adult C57BL6/J mice and maintained under control conditions (5% CO2, balance air) plus thromboxane A2 receptor agonist U46619 (125 nM) to induce a partial constriction in vitro. While monitoring vessel diameter with fluorescent video microscopy, we found that bath application of a selective P2Y2 agonist PSB 1114 constricted arterioles by 10.5% +/− 0.41%, whereas exposure to a selective P2Y2 antagonist AR‐C 118925XX (10 υM) blocked the CO2/H+−evoked vasoconstriction. P2Y2 dependent vasoconstriction in the RTN was also confirmed in vivo in pial vasculature of the RTN. In addition, activation of P2Y2 receptors in the RTN (PSB 1114, 125 nM) evoked an increase in breathing as reported by diaphragmatic EMG. Next, using cell type specific reporter lines (smMHCCre/eGFP, Aldh1l1Cre/TdTomato, Tie2‐Cre/TdTomato), we isolated astrocytes, endothelial cells, and smooth muscle cells from each region of interest (cNTS, ROb, and RTN) for subsequent FACS and qRT‐PCR. Consistent with our hypothesis, we found that P2Y2 mRNA was highly expressed in smooth muscle cells from RTN arterioles but was not detectable in endothelial cells across all regions of interest. P2Y2 mRNA did not appear to be enriched in astrocytes from either region. In addition, calcium imaging experiments show that activation of P2Y2 either pharmacologically (using PSB 1114) or with high CO2 evoked calcium signaling in both penetrating and pial arterioles of the RTN, but not of the cNTS or ROb. Together, these results suggest that specialization of vasculature tone in the RTN is important for contributions in modulating respiratory behavior.Support or Funding InformationNIH/NHLBI F31HL142227, R01HL104101This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.