Purinergic modulation of preBötzinger complex inspiratory rhythm generating networks.

ATP actions on central inspiratory networks are determined by a 3-part signaling system comprising: i) the excitatory actions of ATP at P2 receptors (Rs) ii) ectonucleotidases that degrade ATP into ADP and adenosine (ADO), and iii) the inhibitory actions of ADO at P1Rs. During hypoxia, an initial increase in ventilation is followed by a secondary depression that is life threatening in premature infants. The release of ATP in respiratory networks, including the preBötzinger Complex (preBötC, important site of inspiratory rhythm generation) attenuates this secondary ventilatory depression. The degree of this attenuation, however, will be determined in part by the complement and density of local ectonucleotidases. Degradation of ATP to ADP, a P2YR agonist, could enhance the attenuation while degradation to ADO by different enzymes could exacerbate the depression. My thesis research has explored the significance of this 3-part signaling system for preBötC networks in rodents using primary cultures of preBötC glia, rhythmically-active medullary slices from rats and mice, and anesthetized adult rats. My objective is to use these various reduced preparations to understand the mechanisms by which ATP acts when it is released endogenously within the preBötC. In rhythmic medullary slices from neonatal rats in vitro, injection of ATP into the preBötC evokes a 2-4 fold increase in frequency via a P2Y1R mechanism that involves both neurons and glia. Analysis of cultured preBötC astrocytes suggests that the glial mechanism is mediated through phospholipase C, the release of Ca2+ from intracellular stores and the release of excitatory gliotransmitters, including glutamate, that in turn excite inspiratory neurons. In contrast to rats, however, injection of ATP into the preBötC of rhythmic slices from neonatal mice has no effect on rhythm. However, ATP does evoke a P2Y1R-mediated frequency increase if A1 ADORs are blocked, suggesting that in mice ATP is rapidly broken down into ADO, which acts via A1 ADORs to counteract the excitatory actions of ATP at P2Y1Rs. Consistent with this, ADO inhibits preBötC frequency in mice but not rats. To assess whether differential expression of ectonucleotidases might also contribute to the varied ATP responses in rats vs mice, we analyzed mRNA from preBötC punches using real time PCR to reveal that TNAP, an enzyme that degrades ATP to ADO, is the main ectonucleotidase in mice but NTPDase2, which degrades ATP to ADP (a P2Y1R agonist) is the main ectonucleotidase in rats. The significance of these purinergic signaling mechanisms for mature preBötC networks in vivo was examined in three ways. First, injection of a P2Y1R agonist into the preBötC of urethane anesthetized rats evoked a 40% increase in respiratory frequency. Second, comparing hypoxic ventilatory responses of urethane anesthetized rats revealed that compared to control saline injections, the hypoxic ventilatory depression was much greater following unilateral injection of a P2Y1R antagonist into the preBötC. Finally, the importance of endogenous ATP in establishing the dynamics of the hypoxic ventilatory response was examined by increasing ectonucleotidase activity in the preBötC via local injection of a lentivirus that caused increased expression of the enzyme TMPAP. Compared to rats injected with a control virus, TMPAP-injected animals with elevated ectonucleotidase activity showed a much greater secondary hypoxic ventilatory depression. Data indicate that the effects of ATP on the preBötC network of neonatal rodents are determined by a delicate balance between the excitatory actions of ATP and ADP at P2Rs and the inhibitory actions of P1R actions (on neurons and glia), and that the local subtype and concentration of ectonucleotidase is a key factor in setting this balance. In neonatal rat, the balance favours excitation. In mouse, (at least under conditions of exogenous ATP application) the excitatory actions of ATP and inhibitory actions of ADO are balanced, providing a valuable model to explore how purinergic signaling might be manipulated to tip the balance in favour of excitation to counteract respiratory instabilities as seen in apnea of prematurity. The same appears to apply to adult preBötC networks under conditions of endogenous ATP release during hypoxia where disruption the balance by increasing ectonucleotidase expression significantly alters response dynamics. [ABSTRACT FROM AUTHOR]