Developmental Changes in the Role of Adenosine Clearance Mechanisms in Modulating the Activity of the preBötzinger Complex Inspiratory Network Under Basal and Hypoxic Conditions

Abstract

Exposure to low oxygen (hypoxia) evokes a biphasic hypoxic ventilatory response (HVR) characterized by a carotid‐body‐mediated increase in ventilation followed by a centrally‐mediated secondary hypoxic respiratory depression (HRD). The HRD is strongest and life‐threatening in premature infants (e.g., Apnea of Prematurity). Purinergic signaling plays a prominent role in shaping the HVR; hypoxia evokes release of ATP from astrocytes in the preBötzinger Complex (preBötC, key site of inspiratory rhythm generation), which increases breathing and attenuates the HRD. However, ATP is metabolized into adenosine (ADO), which inhibits the preBötC and is implicated in the HRD. Thus, the net effect of ATP in the preBötC during hypoxia is determined by the balance between the actions of ATP and ADO. The objective of this study is to determine during development how mechanisms important in clearing extracellular ADO (ADOe) influence preBötC activity and the HVR. First, we focused on equilibrative nucleoside transporters (ENTs) that passively move ADO across cell membranes down its concentration gradient. We compared the HVR (10 min 10% O2) of WT and ENT1/2 double KO mice (P0–3, P6–8, and P12–14) using whole‐body plethysmography. The HRD of ENT KO mice was significantly greater compared to WT mice at all ages, reflecting greater reductions in breathing frequency and a likely role for ENTs in removing ADOe during hypoxia. We then applied the ENT inhibitor NBMPR (100 μM) to the solution bathing rhythmic, preBötC‐containing medullary slices from neonatal mice to directly assess the influence of ENTs on basal preBötC rhythm. A significant, NBMPR‐mediated, 16% ± 7% (n=5) increase in frequency suggests that ENTs elevate basal ADOe tone, causing a modest reduction in frequency. In contrast, the reduction in inspiratory frequency evoked by injecting ADOe into the preBötC (to simulate a high ADOe load as may occur in hypoxia) was almost 2‐fold greater in slices from ENT1/2 KO compared to WT mice; i.e., under high load, ENTs appear to clear ADOe. We next examined ADO kinase (ADK), an enzyme that converts ADO into AMP, lowering intracellular ADO levels and facilitating clearance of ADOe by ENTs. Inhibition of ADK with ABT‐702 (10 μM) produced an adenosine‐mediated (i.e., the effect was reversed with the A1R antagonist, DPCPX, 100 nM) decrease in the baseline frequency of rhythmic medullary slices that was 16% ± 2% (n=7) at P0–2 and 24% ± 5% (n=8) by P9–11.In summary, these data suggest that under basal conditions ENTs and ADK are important in controlling ADOe tone, and that under conditions of high ADOe load, as produced here via local injection of ADO in vitro or hypoxia in vivo, ENTs and ADK become important in clearing ADOe, which increases ventilation. Finally, the observation in vitro that ADK activity in the preBötC appears to increase during development raises the intriguing possibility that the greater HRD of premature infants is due, at least in part, to an immature system for clearing ADOe.Support or Funding InformationCIHR, NSERC, WCHRI, AIHS, CFI, Alberta Lung AssociationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.