Postnatal Development of the Purinergic Signaling System in the preBötzinger Complex; Implications for the Hypoxic Ventilatory Response

Abstract

The hypoxic ventilatory response (HVR) comprises a carotid body‐mediated increase in ventilation followed by a secondary, CNS‐mediated decrease, the hypoxic respiratory depression (HRD), that is larger and potentially life‐threatening in newborn mammals. The mechanisms responsible for the greater HRD in newborns are unknown, but purinergic signaling via P2 and P1 receptor (R) mechanisms helps shape this 2° phase. Shortly after the 1° increase in ventilation, astrocytes release ATP in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation), which stimulates breathing via P2Y1Rs and attenuates the HRD. Extracellular ATP (ATPe) is degraded to adenosine (ADOe), which inhibits breathing and is implicated in the HRD. Thus, the impact of ATP on the HVR is determined by the balance between the actions of ATP and ADOe, that could change developmentally and contribute to the greater HRD of newborns. Our goal was to characterize how the many factors that determine the balance between ATP and ADOe signaling change during development, including: P2Y1Rs; ADO A1Rs; ectonucleotidases (ECTOs) that degrade ATPe to ADOe; ADOe clearance by equilibrative nucleoside transporters (ENTs) via transmembrane transport of ADO down its concentration gradient; and ADO kinase (ADK), which is not mature in the cortex until postnatal day (P) 14, but facilitates ADOe clearance by converting ADOi into AMP so that ADOi<ADOe. We used rhythmic preBötC slice preparations to characterize developmental changes in: i) the effects of P2Y1R and ADOR signaling on preBötC rhythm; ii) ECTO activity; and iii) the impact of ENTs and ADK on baseline preBötC rhythm and preBötC responses to ATP and ADO that were locally injected to simulate hypoxia. iv) In vivo head‐out plethysmography was used to assess the role of ADOe clearance mechanisms in shaping the developing HVR.i) The frequency (freq) increase evoked by ATP in the preBötC doubled between P0‐6 and P9‐12, but the P2Y1R agonist MRS 2365‐evoked increase did not change. The A1R inhibition of freq was of similar magnitude but greater duration at P0‐6 compared to P9‐12. ii) ECTO activity, assessed by incubating preBötC tissue in ATP and measuring PO4, was greater at P9‐12. iii) The ADO freq inhibition was 2‐fold longer in ENT knockout mice compared to WT mice at all ages. ADK inhibition (ABT 702) inhibited baseline freq only at P9‐12. iv) Knockout of ENTs blunted the freq response to hypoxia (10% O2) in vivo, but increased the VT response at all ages. Introduction of constitutively active ADK into the brain produced P0‐2 ADKtg mice that responded to hypoxia with an adult‐like, sustained increase in VE/VO2; P0‐2 WT C57BL6 did not respond to hypoxia. This difference disappeared by P6‐8. Surprisingly, the mature HVR of P0‐2 ADKtg mice reflected a greater hypoxic depression of VO2, not a greater increase in VE.Data suggest preBötC sensitivity to P2Y1 and A1Rs does not change, while ECTO activity increases developmentally. ADO actions are prolonged in P0‐6 mice, suggesting that ADOe clearance mechanisms mature postnatally. ENTs appear to play a similar role from birth, which may involve clearing ADOe from the preBötC during hypoxia. ADK maturation also appears important in the development of an adaptive HVR. However, the role of ADK in modulating VE vs metabolic responses to hypoxia remains to be elucidated.