The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response

Abstract

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.

Keywords: P1 receptor; P2 receptor; adenosine kinase; ectonucleotidase; equilibrative nucleoside transporter; hypoxia.

FIGURE 1

The effects of ATP released into the extracellular space is determined by a balance between actions of ATP at P2 receptors and ADO, its main metabolite, at P1 receptors. Extracellular ATP has two main fates, binding to P2 receptors (1) or degradation by diverse ectonucleotidases (ECTOs) (2) with differential substrate affinities that degrade ATP and its by-products ultimately to adenosine (ADO). Extracellular ADO has three main fates, binding to P1 receptors, transport into the intracellular space or remaining where it is. The balance is dynamic and determined by a complex system referred to as the purinome (see Figure 2) (Modified with permission from Funk, 2013).

FIGURE 2

Simplified schematic of the purinome at a glutamatergic, inspiratory, preBötC synapse: multiple factors determine the balance between ATP and ADO signaling. During inspiration, a volley of action potentials in the presynaptic neuron evokes glutamate release that depolarizes the postsynaptic inspiratory neuron via activation of glutamate receptors (ionotropic and metabotropic, mGluR). Hypoxia stimulates astrocytes, via a mitochondrial mechanism (not shown) that evokes increased intracellular Ca²⁺ and vesicular ATP release (1). (2) ATPe acts via neuronal P2 (primarily P2Y₁) receptors to excite inspiratory neurons and increase ventilation via a process involving increased intracellular Ca²⁺. Extracellular ADO (ADOe) increases through breakdown of ATPe by ectonucleotidases (3) or ENT transport of accumulating ADOi (5). ADO acts pre- and postsynaptically via A1 receptors (or A2 receptors on GABAergic neurons, not shown) to inhibit ventilation (4). The direction of ADO transport via ENTs (5) is dependent on the [ADO] gradient. Hypoxia also causes accumulation of intracellular ADO (ADOi) from ATPi hydrolysis (6). ADK phosphorylates ADO into AMP (7), keeping ADOi low so that ENTs remove ADOe. The cytoplasmic form of ADK, at least in adult brain, is limited to astrocytes so that removal of ADOe becomes an astrocyte dependent process (Modified with permission from Funk, 2013).

FIGURE 3

Differential balance between the actions of ATP and ADO in rhythmically-active preBötC-containing slices from neonatal rat and mouse. Integrated XII nerve recordings (∫XII) from rhythmic slices of rat and mice showing baseline inspiratory-related rhythm in vitro and responses to local injection of ATP (A) or the P2Y₁ receptor agonist, MRS-2365, into the preBötC (B). (C) Response of a mouse slice to ATP under control conditions and after preBötC injection of the A1 receptor antagonist DPCPX. (D) Real-time PCR analysis showing the percentage contribution of each ectonucleotidase isoform to the total ectonucleotidase mRNA extracted from mouse and rat preBötC punches. Error bars indicate SEM. ^∗Significant difference between the compared columns. Reproduced with permission from Zwicker et al. (2011).

FIGURE 4

Global knock out of ENTs increases the secondary hypoxic respiratory depression. (A) Cartoon showing the biphasic hypoxic ventilatory response, which comprises a Phase 1 increase followed by a secondary depression to a lower, steady-state level of ventilation in Phase 2. The Phase 1 increase was reported as 100%. Ventilation during Phase 2 was calculated as a percentage of the peak increase during Phase 1. In this mock example, ventilation during phase 2 was ∼45% of the phase 1 increase; i.e., ventilation fell by ∼55% from the peak during the hypoxic respiratory depression. (B) The level of ventilation (measured using whole-body plethysmography) during Phase 2 of the hypoxic ventilatory response is reported relative to the Phase 1 peak increase for unanesthetized wild type (WT), ENT2, ENT1, and ENT1/2 double knockout mice. ^∗Represents sig. difference from WT, p < 0.05 or ^∗∗p < 0.01. ANOVA was used in conjunction with Bonferroni post hoc multiple comparison test.