Modulation of synaptic and channel activities in the respiratory network of the mice by NO/cGMP signalling pathways

Abstract

We examined signalling pathways which can involve NO as a second messenger in the respiratory network. In the functional slice preparation, NO donors depressed the respiratory motor output and enhanced its depression after brief episodes of hypoxia. In the inspiratory neurons, NO donors suppressed spontaneous excitatory and inhibitory synaptic currents, activated single K_ATP channels and inhibited L-type Ca²⁺ channels. NO scavengers, PTIO and hemoglobin, and the blocker of NO synthase, N-monomethyl-l-arginine, induced effects opposite to those of NO donors and indicated the role of endogenously generated NO in the modulation of the respiratory activity. Using fluorescent dyes DAF-2 and DCF, we imaged NO and reactive oxygen species (ROS). Concentrations of NO and ROS increased during brief episodes of hypoxia and they both contributed to the activation of K_ATP channels due to oxygen withdrawal. The oxidizing agent t-butyl-hydroperoxide acted similarly to NO donors but it did not interfere with the effects of NO. Increase in cGMP levels with 8-Br-cGMP reproduced the actions of NO donors and occluded the effects of their subsequent applications. We propose that in the respiratory neurons, a constitutive production of NO is responsible for a tonic activation of cGMP-coupled signalling pathways and changes in NO levels modulate the respiratory motor output by altering the activity of K_ATP and L-type Ca²⁺ channels.

Introduction

Nitric oxide, a membrane-permeant messenger in various physiological and pathological processes, acts as a local neuromodulator in the CNS (cf. Prast and Philippu, 2001, Keynes and Garthwaite, 2004, Calabresi et al., 2000). NO modifies electrical activity via NO/cGMP pathway and reversible oxidization of sulfhydryl (–SH) groups of corresponding channels and receptors (Jacintho and Kovacic, 2003, Fischmeister et al., 2005, Xu et al., 2004). NO synthases (NOS) are responsible for constitutive production of NO and it can be additionally enhanced during ischemia and hypoxia due to disturbances in energy metabolism (Bolanos and Almeida, 1999, Keynes and Garthwaite, 2004). In the heart, several ion channels have been shown to be modified by NO, such as L-type Ca²⁺ (Ca_L), ATP-sensitive (K_ATP), and pacemaker f-channels (Fischmeister et al., 2005) that is mediated by cGMP. K_ATP channels and NOS activity are important in cardioprotection (Xu et al., 2004) and neuroprotection (Bolanos and Almeida, 1999, Moncada and Bolanos, 2006) that also involves the activation of NO-cGMP-PKG signalling pathway.

Whole-animal studies have shown that NO markedly affects sympathetic outflow, alter respiratory rhythm and influence pain thresholds in the spinal cord. The activation of the NO/cGMP pathway causes direct blockade of acute and persistent hypernociception by opening K_ATP channels via stimulation of PKG (Sachs et al., 2004). Sensory neurons respond to hypoxia with an activation of NOS due to Ca²⁺ influx via Ca_L channels that results in enhanced NO production which is associated with mitochondria and contributes to resistance against hypoxia (Henrich et al., 2004). Nitric oxide acts as a retrograde messenger in the nucleus tractus solitarii (NTS) (Ogawa et al., 1995) where a reciprocal regulation of NO and glutamate exists (Lin et al., 2000). If similar signalling pathways are expressed in the respiratory network which is located ventral to NTS, this would modulate the respiratory motor output driven by glutamatergic interneurons which are also involved in the actions of hypoxia on breathing (Kline et al., 1998). In the rat and the cat brainstem, chronic pretreatment of rats with NOS blockers causes a significant decrease in cGMP, and attenuates the ventilatory response to hypoxia (Haxhiu et al., 1995) which is consistent with enhanced NO production during hypoxia as observed in other brain regions (Bolanos and Almeida, 1999, Keynes and Garthwaite, 2004).

NO actions in respiratory neurons have not been yet investigated. We here report that NO donors and NOS inhibitors modulated the respiratory motor output in the functional slice preparation. The two classes of drugs produced opposite effects. The modulation of the respiration-related variables was accompanied by changes in the amplitude but not in the frequency of spontaneous synaptic currents that indicated postsynaptic mechanisms of NO actions. In the presence of NO donors and NOS inhibitors, the response of the respiratory network to hypoxia was also modified that involved modulation of the activity of K_ATP and Ca_L channels in the inspiratory neurons. Using DAF-2 and carboxy-H₂CFDA (DCF), the dyes which respectively sense NO and reactive oxygen species (ROS), we observed a localized production of both moieties in the vicinity of mitochondria which was enhanced during hypoxia. The actions of NO on synaptic and ion channel activities did not interfere with the effects of ROS. Instead, all observed responses which involved NO were reproduced by elevations of intracellular cGMP levels. Thus, regulation of respiratory rhythmogenesis by NO/cGMP complements previously described mechanisms of respiratory rhythm modulation utilizing protein kinases A and C, and G-proteins (Haji et al., 1996, Lalley et al., 1997, Johnson et al., 1996, Pierrefiche et al., 1996, Mironov and Richter, 2000a, Mironov and Richter, 2000b).