Molecular mechanisms involved in P2Y1 receptor-mediated excitation of the inspiratory rhythm generating network

• Author / Creator

  Jalubula, Venkatesh

• Hypoxic ventilatory response is biphasic, comprising of an initial increase in ventilation followed by secondary depression. Initial increase in ventilation is mediated by peripheral carotid bodies where as central mechanisms were implicated in secondary depression. The secondary depression phase is more pronounced in premature infants which is life threatening. P2Y1 receptors play an important role in shaping hypoxic ventilatory response. During hypoxia ATP is released in the ventral respiratory column, including the preBötzinger Complex (preBötC, key site of inspiratory rhythm generation), where it evokes a P2Y1 receptor mediated increase in inspiratory frequency that attenuates the secondary depression. The molecular mechanisms involved in the network excitation by ATP are unknown. In other parts of the brain, P2Y1 receptors are coupled to the Gαq-signaling pathway, which involves phospholipase C and inositol trisphosphate-mediated release of Ca2+ from intracellular stores. Thus, I predicted that preBötC inspiratory neurons would respond to P2Y1 receptors activation with an increase in Ca2+ fluorescence. 18 inspiratory neurons from 5 slices showed a ~50% increase in baseline fluorescence to local applied P2Y1 receptor agonist MRS 2365 (100µm, 10 Sec). Based on the high sensitivity of the network to P2Y1R activation, I hypothesized that a population of neurons that is important in rhythm generation or in providing direct excitatory drive to rhythm generating neurons, are very sensitive to P2Y1 receptor activation. I tested the hypothesis that not all Inspiratory neurons are equally sensitive to MRS 2365 evoked increase in Ca2+i. Responders, whose fluorescence change was obvious on visual inspection, constituted 12 neurons of 20 neurons screened. A detailed quantification revealed 47 ± 10% and 6.5 ± 2.8% increase of their baseline Ca2+ fluorescence in responders and non-responders respectively. I next tested the source of MRS 2365 evoked Ca2+ and its contribution to respiratory network excitation. Depleting Ca2+i with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) blockers attenuated the MRS 2365-evoked increase in Ca2+i by 44 ± 7%. At network level SERCA blockers thapsigargin (200µM) and cyclopiazonic acid (CPA) attenuated the MRS 2365 evoked frequency increase by 27 ± 7% and 32 ± 8% respectively. I next screened G protein-coupled inwardly-rectifying potassium channel (GIRK; BaCl2), SK (Apamin), Voltage gated K+ channels (Tetraethylammonium) (TEA), type I BK (Iberiotoxin) channels as downstream effector of P2Y1 receptor and found no effect on MRS 2365 evoked frequency increase, while Paxilline, a type I & II BK channel blocker at 1 µM attenuated MRS 2365 evoked frequency increase by 29 ± 13% suggesting a possible role of Ca2+i and type II BK channels in P2Y1 receptor mediated network excitation.

• Subjects / Keywords

  • Hypoxia

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3J38KX7D

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.