Identification of the mechanisms through which ATP excites the inspiratory network in vitro

• Author / Creator

  Zhang, Yong

• The ventilatory response to hypoxia comprises an initial peripheral chemoreceptor-mediated increase in ventilation followed by a centrally-mediated secondary depression that can be life-threatening in premature infants with apnea. ATP appears to be released from astrocytes in the preBötzinger Complex (preBötC, critical site for inspiratory rhythm generation), during hypoxia where it attenuates the secondary depression via a P2Y1 receptor-dependent excitation of inspiratory neurons. However, the underlying mechanism(s) coupled to P2Y1 receptors is unknown. Here we apply nerve and whole-cell recording methods to rhythmic medullary slices (700 µm) from neonatal rat to test the hypothesis that the P2Y1R operates through 1) the Gαq-signalling pathway and 2) elevation of cAMP levels and potentiation of the hyperpolarization-activated inward current, Ih, to excite the inspiratory neurons and network. We found that blocking the individual steps of the Gαq-signalling pathway via intracellular dialysis of the second messenger blockers attenuated the 5 mM ATP- or MRS 2365 (P2Y1 receptor agonist, 100 µM)-induced inward currents by 20% - 30% and ~55%, respectively. At the network level, blocking the Gαq-signalling pathway reduced the MRS 2356 (P2Y1 receptor agonist)-induced network excitation by up to ~60%. These data suggest that the Gαq-signalling contributes to about 50 - 60% of the P2Y1 receptor-meditated excitation. We also discovered that the MRS2365 current reversed between -60 and -40 mV, suggesting activation of Ih. ZD7288 (open channel blocker of Ih, 100 µM) attenuated he MRS 2365-induced inward current by ~35% (15 min) and –network excitation by ~60% (1st trial). P2Y1 receptor activation induced an ~9.8 mV depolarizing shift in V1/2 (membrane potential at which 50% of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie Ih, are open). Moreover,blocking cAMP production by SQ 22536 (adenylyl cyclase inhibitor) attenuated the MRS 2365-induced currents by ~60 % (15 min, in 9 of 18 cells) and -network excitation by ~50% (60 min, bath). These data suggest that the P2Y1 receptor-mediated excitation of the preBötC network is produced via 1) activation of the Gαq-signalling pathway, and 2) an elevation in cAMP level and potentiation of Ih in a subpopulation of inspiratory neurons.

• Subjects / Keywords

  • Purinergic signalling
  • P2Y1 receptor
  • Inspiratory neuron
  • Pre-Bötzinger complex
  • Hypoxic ventilatory response

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-qpb6-g836

• License

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