P2Y1 receptor-mediated potentiation of inspiratory motor output in neonatal rat in vitro.

PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y1R) activation. Despite this, and the fact that inspiratory MNs express P2Y1Rs, the role of P2Y1Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y1R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y1R agonist, 0.1 mm) potentiated XII inspiratory burst amplitude by 60 ± 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca(2+)-dependent non-selective cation current ICAN; high intracellular concentrations of BAPTA, which buffers Ca(2+) increases necessary for activation of ICAN; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for ICAN). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y1R modulation than phrenic MNs and that the P2Y1R potentiation of inspiratory output occurs in part via potentiation of TRPM4-mediated ICAN, which amplifies inspiratory inputs.

Adrenergic α1 receptor activation is sufficient, but not necessary for phrenic long-term facilitation.

Acute intermittent hypoxia (AIH; three 5-min hypoxic episodes) causes a form of phrenic motor facilitation (pMF) known as phrenic long-term facilitation (pLTF); pLTF is initiated by spinal activation of Gq protein-coupled 5-HT2 receptors. Because α1 adrenergic receptors are expressed in the phrenic motor nucleus and are also Gq protein-coupled, we hypothesized that α1 receptors are sufficient, but not necessary for AIH-induced pLTF. In anesthetized, paralyzed, and ventilated rats, episodic spinal application of the α1 receptor agonist phenylephrine (PE) elicited dose-dependent pMF (10 and 100 µM, P < 0.05; but not 1 µM). PE-induced pMF was blocked by the α1 receptor antagonist prazosin (1 mM; -20 ± 20% at 60 min, -5 ± 21% at 90 min; n = 6). Although α1 receptor activation is sufficient to induce pMF, it was not necessary for AIH-induced pLTF because intrathecal prazosin (1 mM) did not alter AIH-induced pLTF (56 ± 9% at 60 min, 78 ± 12% at 90 min; n = 9). Intravenous (iv) prazosin (150 µg/kg) appeared to reduce pLTF (21 ± 9% at 60 min, 26 ± 8% at 90 min), but this effect was not significant. Hypoglossal long-term facilitation was unaffected by intrathecal prazosin, but was blocked by iv prazosin (-4 ± 14% at 60 min, -13 ± 18% at 90 min), suggesting different LTF mechanisms in different motor neuron pools. In conclusion, Gq protein-coupled α1 adrenergic receptors evoke pMF, but they are not necessary for AIH-induced pLTF.

Systemic LPS induces spinal inflammatory gene expression and impairs phrenic long-term facilitation following acute intermittent hypoxia.

Although systemic inflammation occurs in most pathological conditions that challenge the neural control of breathing, little is known concerning the impact of inflammation on respiratory motor plasticity. Here, we tested the hypothesis that low-grade systemic inflammation induced by lipopolysaccharide (LPS, 100 µg/kg ip; 3 and 24 h postinjection) elicits spinal inflammatory gene expression and attenuates a form of spinal, respiratory motor plasticity: phrenic long-term facilitation (pLTF) induced by acute intermittent hypoxia (AIH; 3, 5 min hypoxic episodes, 5 min intervals). pLTF was abolished 3 h (vehicle control: 67.1 ± 27.9% baseline; LPS: 3.7 ± 4.2%) and 24 h post-LPS injection (vehicle: 58.3 ± 17.1% baseline; LPS: 3.5 ± 4.3%). Pretreatment with the nonsteroidal anti-inflammatory drug ketoprofen (12.5 mg/kg ip) restored pLTF 24 h post-LPS (55.1 ± 12.3%). LPS increased inflammatory gene expression in the spleen and cervical spinal cord (homogenates and isolated microglia) 3 h postinjection; however, all molecules assessed had returned to baseline by 24 h postinjection. At 3 h post-LPS, cervical spinal iNOS and COX-2 mRNA were differentially increased in microglia and homogenates, suggesting differential contributions from spinal cells. Thus LPS-induced systemic inflammation impairs AIH-induced pLTF, even after measured inflammatory genes returned to normal. Since ketoprofen restores pLTF even without detectable inflammatory gene expression, "downstream" inflammatory molecules most likely impair pLTF. These findings have important implications for many disease states where acute systemic inflammation may undermine the capacity for compensatory respiratory plasticity.

Systemic inflammation impairs respiratory chemoreflexes and plasticity.

Many lung and central nervous system disorders require robust and appropriate physiological responses to assure adequate breathing. Factors undermining the efficacy of ventilatory control will diminish the ability to compensate for pathology, threatening life itself. Although most of these same disorders are associated with systemic and/or neuroinflammation, and inflammation affects neural function, we are only beginning to understand interactions between inflammation and any aspect of ventilatory control (e.g. sensory receptors, rhythm generation, chemoreflexes, plasticity). Here we review available evidence, and present limited new data suggesting that systemic (or neural) inflammation impairs two key elements of ventilatory control: chemoreflexes and respiratory motor (versus sensory) plasticity. Achieving an understanding of mechanisms whereby inflammation undermines ventilatory control is fundamental since inflammation may diminish the capacity for natural, compensatory responses during pathological states, and the ability to harness respiratory plasticity as a therapeutic strategy in the treatment of devastating breathing disorders, such as during cervical spinal injury or motor neuron disease.

ATP in central respiratory control: a three-part signaling system.

The landmark demonstrations in 2005 that ATP released centrally during hypoxia and hypercapnia contributes to the respective ventilatory responses validated a decade-old hypothesis and ignited interest in the potential significance of P2 receptor signaling in central respiratory control. Our objective in this review is to provide a non-specialist overview of ATP signaling from the perspective that it is a three-part system where the net effects are determined by an interaction between the signaling actions of ATP and adenosine at P2 and P1 receptors, respectively, and a family of enzymes (ectonucleotidases) that breakdown ATP into adenosine. We review the rationale for the original interest in P2 signaling in respiratory control, the evolution of this hypothesis, and the mechanisms by which ATP might affect respiratory behaviour. The potential significance of P2 receptor, P1 receptor and ectonucleotidase diversity for the different compartments of the respiratory control system is also considered. We conclude with a look to future questions and technical challenges.