Limited Capacity for Clearance of Adenosine from the preBötzinger Complex Inspiratory Network Contributes to the Profound Depression of Breathing by Hypoxia in the Very Young

Abstract

Breathing in premature infants often stops briefly (apnea) because the brainstem network that controls breathing is immature (apnea of prematurity, AOP). Apneas cause hypoxia, triggering the hypoxic ventilatory response (HVR), which comprises an initial increase in ventilation followed by a centrally mediated 2° decrease. In adults, breathing remains above baseline during this 2° phase, but in the very young it falls below baseline leading to a life‐threatening feedback loop where apnea causes hypoxia, respiratory depression, further hypoxia and so on. Understanding the cause of this powerful hypoxic inhibition of breathing in the very young, a major question in perinatal physiology, could inform new therapies for AOP. The transmitter ATP is released in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation) during hypoxia where it increases breathing and attenuates the 2° depression. However, extracellular ATP (ATPe) is degraded to adenosine (ADO), which inhibits breathing and is implicated in AOP; e.g., caffeine blocks ADOe actions and is the first line treatment for AOP. We hypothesize that the greater 2° inhibition of breathing in prematurity is due to an immature system for clearing ADOe. ADOe‐mediated inhibition stops with its removal from the extracellular space, which depends on i) equilibrative nucleoside transporters (ENTs) that move ADOe across cell membranes down its concentration gradient, and ii) ADO kinase (ADK), an enzyme that keeps the level of ADO inside cells lower than outside so ENTs clear ADOe. How ENT activity develops in the preBötC is unknown. However, the form of ADK that influences ADOe in the brain is minimally functional until 2 weeks of age in rodents. To assess the importance of ADOe clearance in the development of the HVR, we used plethysmography to measure the HVR (10% O2) in 0‐56 day old (P0‐14) wild‐type (WT), ENT knockout (KO), and ADKtg mice engineered to have functional ADK throughout life. In WT and ENT KO neonates (P0‐2), ventilation fell below baseline after ~7 min in hypoxia, but the hypoxia‐evoked frequency increase was markedly reduced in the ENT KO. Strikingly, P0‐2 ADKtg mice showed the mature breathing response; ventilation remained above baseline levels throughout hypoxia. At P12 in WT and ENT KO, the HVR had still not matured; ventilation fell below baseline during the 2° phase, yet it remained above baseline in P12 ADKtg mice. To assess the importance of ENT and ADK specifically in the preBötC, we isolated the preBötC network in rhythmic brain slices from P0‐12 mice. The ADO inhibition of preBötC frequency lasted twice as long in P0‐12 ENT KO mice compared to WT. Conversely, the decrease in basal frequency caused by ADK inhibition (ABT‐702) increased with age (P0‐2, 15%; P9‐11, 23%). These data suggest that i) ENTs are important for ADO clearance in the preBötC after birth and ii) low ADK activity at birth is a major contributor to the greater hypoxic respiratory depression in early development.