Impact of Equilibrative Nucleoside (Adenosine) Transporters on the preBötzinger Complex Inspiratory Network and the Hypoxic Ventilatory Response in Newborn Mice

Abstract

Exposure to low oxygen (hypoxia) evokes a biphasic hypoxic ventilatory response (HVR) comprising of an initial increase in ventilation followed by a secondary depression. The secondary depression is much stronger and life‐threatening in premature infants and it is implicated in apneic events (Apnea of Prematurity). Despite the clinical significance, mechanisms underlying this secondary depression are poorly understood. Recent data suggest that during hypoxia, astrocytes in the preBötzinger complex (preBötC) release ATP, which increases breathing and attenuates the secondary depression. However, ATP is rapidly metabolized into extracellular adenosine (ADOe), which is implicated in the secondary depression. Thus, the overall effects of ATP are determined by a balance between its excitatory actions and the inhibitory actions of its by‐product, ADO. The importance of ADO is clear in that infants with apnea of prematurity are given caffeine (ADO receptor antagonist) to stimulate breathing. The ATP‐ADO balance is controlled by many factors, including equilibrative nucleoside transporters (ENTs) that move ADO across cell membranes along its concentration gradient. Our goal was to determine the role of ENTs in modulating preBötC network activity under baseline and hypoxic conditions. Bath application of the ENT‐1 inhibitor (NBMPR, 100 μM) to rhythmically‐active medullary slices from neonatal mice caused a significant 9.9±4% increase in frequency (n=5). However, when the preBötC was challenged with an ADO load (as might happen during hypoxia) via local injection of ADO (500 μM, 30 s) ENT‐1 inhibition enhanced the inhibitory effect. Similarly, when we compared the responses of wild type (WT) and ENT 1/2 knockout (ENT KO) mice to preBötC ADO injections, the duration of the frequency inhibition was almost doubled in the KO mice. We next compared the HVR of WT and ENT KO mice (1–3 days old) using whole‐body plethysmography (15 min control, 10 min 10% O2, 10 min recovery). Ventilation increased in both groups by 20% and gradually fell below baseline in WT mice after 4 min in hypoxia. In marked contrast, ventilation fell below baseline during the first 40 s of hypoxia in ENT KO mice by as much as 25% and never increased above baseline throughout the hypoxic challenge. These in vitro data suggest that ENTs contribute to setting ADOe tone in the preBötC under baseline conditions by transporting ADO into the extracellular space. However, under an extra load as might occur in hypoxia, ENTs become important in removing ADOe. In vivo data are consistent with this hypothesis, but it will be critical to determine whether the differences in the HVR of WT and ENT KO mice in vivo reflects loss of ENT activity in the carotid bodies or central nervous system.Support or Funding InformationCIHR, NSERC, WCHRI, AIHS, CFIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.