Vascular control of the CO2/H+-dependent drive to breathe

  1. Colin M Cleary
  2. Thiago S Moreira
  3. Ana C Takakura
  4. Mark T Nelson
  5. Thomas A Longden
  6. Daniel K Mulkey

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Abstract

Respiratory chemoreceptors regulate breathing in response to changes in tissue CO 2 /H + . Blood flow is a fundamental determinant of tissue CO 2 /H + , yet little is known regarding how regulation of vascular tone in chemoreceptor regions contributes to respiratory behavior. Previously, we showed in rat that CO 2 /H + -vasoconstriction in the retrotrapezoid nucleus (RTN) supports chemoreception by a purinergic-dependent mechanism (Hawkins et al., 2017). Here, we show in mice that CO 2 /H + dilates arterioles in other chemoreceptor regions, thus demonstrating CO 2 /H + vascular reactivity in the RTN is unique. We also identify P2Y 2 receptors in RTN smooth muscle cells as the substrate responsible for this response. Specifically, pharmacological blockade or genetic deletion of P2Y 2 from smooth muscle cells blunted the ventilatory response to CO 2 , and re-expression of P2Y 2 receptors only in RTN smooth muscle cells fully rescued the CO 2 /H + chemoreflex. These results identify P2Y 2 receptors in RTN smooth muscle cells as requisite determinants of respiratory chemoreception.

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  1. Version published to 10.7554/elife.59499 on eLife
  2. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on July 9 2020, follows. The preprint has been revised in response to the comments below.

    Summary

    This paper is an important extension of the authors' previous publication in eLife (Hawkins et al. 2017) that presented novel data suggesting that CO2/H+-mediated vasoconstriction in the brainstem retrotrapezoid nucleus (RTN) supports chemoreception by a purinergic-dependent mechanism. Here the investigators provide new data indicating that CO2/H+ dilates arterioles in other chemoreceptor regions (cNTS, raphe obscurus- ROb), thus suggesting that the CO2/H+ vascular reactivity in the RTN is unique compared to some other brain regions. The investigators significantly advance their previous work by applying a number of new experimental approaches to provide evidence that P2Y2 receptors in RTN vascular smooth muscle cells are responsible for the purinergic mechanism mediating the vascular reactivity and specifically contribute to RTN chemosensitivity. Importantly, pharmacological blockade or genetic deletion of P2Y2 from smooth muscle cells blunted the in vivo ventilatory response to CO2, and virally-driven re-expression of P2Y2 receptors in RTN smooth muscle cells rescued the ventilatory response to CO2, suggesting that these receptors are required for the normal ventilatory response to CO2. New pharmacological evidence is also presented that activation of RTN astrocytes is involved in purinergic signaling driving the RTN vasomotor responses. Overall these results advance the concept that specialized vasoreactivity to CO2/H+ in the RTN contributes to respiratory chemoreception.

    Essential Revisions

    1. Although authors are given leeway in the format of a Research Advance, this paper would benefit from more structure including delineation of Introduction, Results, and Discussion sections. The manuscript would be substantially improved in particular by including a more thorough, dedicated Discussion section with explicit elaboration on limitations of their experimental methods and conclusions, and including discussion of how the important P2Y2 receptor knockout and re-expression experiments represent a fundamental advance considering that the authors had already implicated (although not completely established) these receptors in their previous publication.

    2. Presentation of the RT-PCR data of purinergic receptor expression profiles can be improved, particularly by providing a more convincing validation of this data such as giving supplemental data of raw numbers for GAPDH levels across areas to prove that GAPDH actually is a valid reference. The authors could also use 3-4 such genes as many investigators do for expression profile calibration. The reviewers note that for the argument it is not necessarily that important how the levels of receptors look in relation to a house keeping gene, but whether P2Y2 is the only receptor which is relatively highly expressed in RTN smooth muscle cells compared to other regions. Looking at Fig. 1B, it seems that relative to the two other areas, P2X1, P2X4 and P2Y14 are also much higher in RTN smooth muscle cells compared to NTS. The reviewers agree that an important aspect is the remarkably low expression of P2Y2 in endothelium which in theory should oppose constriction by possibly releasing NO.

    3. Additional information on measurements of vascular diameters would be useful. Have the authors obtained measurements from multiple vessels at each time point in the chosen field(s) of view for individual experiments? If so, how do such measurements compare to the representative single vessel measurements for a given experiment presented in the figures? How many vessels per experiment are included in the group summary data? Please explain more completely why it was necessary to induce a 20-30% vasoconstriction by the thromboxane A2 receptor agonist before the measurements.

    4. Some additional validation of the specificity of the AAV2 used for the P2Y2 re-expression experiments would be helpful since this is not a well characterized virus and may lead to receptor overexpression. Additional nice clear images with proper co-localization would be good to see and additional details about non-smooth muscle cell expression should be provided.

    5. The experiments showing unstable breathing in vivo produced by injecting a thromboxane A2 receptor agonist vasoconstrictor (U46119) into the cNTS and ROb under conditions of mild hypercapnia (2-3% inspired CO2) are intriguing, but these experiments lack the proper control of U46119 injections into the cNTS and ROb under normocapnic conditions to determine if this alters blood pressure and produces breathing instabilities independent of any "gain-up" of RTN activity. It would also be of interest to know whether the authors have tested if larger instabilities occur with cNTS/ROb vasoconstriction at higher levels of hypercapnia.

  3. Version published to 10.1101/2020.08.19.257386 on bioRxiv