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

Abstract

Key pointsr The role of metabotropic purinergic receptors (P2YRs) in modulating motor output from the CNS is virtually unknown, despite the fact that many motoneurons, including respiratory motoneurons, express P2YRs.r Using rhythmically active brainstem-spinal cord and medullary slice preparations, we demonstrate that compared to the 4th cervical spinal nerve (C4) inspiratory output controlling the diaphragm, P2YR activation is >10 times more efficacious at potentiating the hypoglossal nerve (XII) inspiratory output controlling airway muscles.r P2YR potentiation of inspiratory output appears largely mediated by P2Y 1 R. r Whole-cell recordings from XII motoneurons (MNs) suggest that the P2Y 1 R-mediated potentiation of inspiratory synaptic inputs, glutamate currents, and persistent inward currents, results in part from potentiation of a transient receptor potential cation channel, subfamily M, member 4 (TRPM4)-mediated, calcium-activated, non-specific cation current, I CAN .r The low sensitivity of phrenic output to P2YR activation questions its physiological significance in modulating diaphragm activity. However, the greater sensitivity of XII MNs, combined with observations that ATP is often co-released with noradrenaline and that noradrenergic neuron activity decreases in sleep, makes it tempting to speculate that loss of purinergic modulation contributes to state-dependent reductions in XII MN excitability.Abstract PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y 1 R) activation. Despite this, and the fact that inspiratory MNs express P2Y 1 Rs, the role of P2Y 1 Rs 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 P2Y 1 R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y 1 R 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 I CAN ; high intracellular concentrations of BAPTA, which buffers Ca 2+ increases necessary for activation of I CAN ; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for I CAN ). 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 P2Y 1 R modulation than p...

O bloqueio purinérgico no núcleo retrotrapezóide (RTN) atenua as respostas respiratórias promovidas pela ativação dos quimiorreflexos central e periférico em ratos.

“…Therefore, we believe that ATP will be involved in the following chain of events: carotid body activation by low levels of O2 stimulates carotid sinus nerve terminals by sending information to the second-order neurons into the NTS; these second-order neurons will activate the respiratory neurons located in the ventral respiratory group (Alvares et al, 2014. We also showed that the P2Y1 blocker did not change the ventilatory response to hypoxia in awake unrestrained rats.…”

“…Há evidências emergentes de que os astrócitos são a fonte do componente purinérgico para os neurônios quimiossensíveis do RTN qual reduziu a resposta respiratória frente à hipóxia . Ainda, outros estudos mostram a participação purinérgica na coluna respiratória dorsal e ventral durante a hipóxia (ALVARES et al, 2014;BRACCIALLI et al, 2008;FUNK et al, 2015); no entanto pouco se sabe especificamente da participação do componente purinérgico no RTN no quimiorreflexo periférico. Após a cirurgia encefálica, os ratos receberam uma injeção intramuscular (0,2 ml/rato) de Pentabiótico Veterinário -Pequeno Porte (Fort Dodge Saúde Animal Ltda.)…”

“…Nossos resultados corroboram com estudos em camundongos knockout para os receptores P2X 2 , que mostraram uma depressão respiratória durante hipóxia -MENDONCA et al, 2009;ALVARES et al, 2014;BRACCIALLI et al, 2008;BRAGA et al, 2007; NURSE, 2014;ZHANG et al, 2012).…”

“…Por outro lado, experimentos do nosso laboratório têm mostrado mediante a realização de experimentos in vitro e in vivo (animais anestesiados), que os neurônios do RTN são altamente sensíveis ao pH e que a sinalização purinérgica contribui para apenas uma parte das respostas ventilatórias promovidas pelas alterações dos níveis de CO 2 /H + qual reduziu a resposta respiratória frente à hipóxia . Ainda, outros estudos mostram a participação purinérgica na coluna respiratória dorsal e ventral durante a hipóxia (ALVARES et al, 2014;BRACCIALLI et al, 2008;FUNK et al, 2015); no entanto pouco se sabe especificamente da participação do componente purinérgico no RTN no quimiorreflexo periférico. http//rsb.info.nih.gov/ij/), que permitiu as contagens de neurônios unicamente e duplamente marcados na região do RTN (Bregma: -11,9 a -11,0).…”

“…10) Nos últimos anos, a sinalização purinérgica tem sido proposta para ser um excelente alvo para terapias de diversas patologias, principalmente devido a novos agentes farmacológicos a serem desenvolvidos . Por outro lado, um passo essencial para o controle da respiração frente a hipercapnia é a ativação dos neurônios do RTN, via sinalização purinérgica, que por sua vez se projeta para a coluna respiratória ventral via sinais excitatórios (ALVARES et al, 2014;. Este mecanismo ocorre também pela participação dos astrócitos, liberando o ATP no RTN pelo processo de exocitose dependente de cálcio o qual é desencadeado pela acidificação intracelular e/ou transporte de CO 2 através de canais do tipo conexinas .…”