NADPH oxidase activity is necessary for acute intermittent hypoxia‐induced phrenic long‐term facilitation

Abstract

Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O 2 , 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (∼C 4 ) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C 4 spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91 phox , whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47 phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C 4 ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.

Repetitive acute intermittent hypoxia increases expression of proteins associated with plasticity in the phrenic motor nucleus

“…the “Q pathway;” Dale-Nagle et al, 2010) include greater serotonin terminal density, and 5-HT 2A receptor, BDNF, TrkB and phospho-ERK expression. We anticipate interesting changes in the expression of other proteins known to play key roles in AIH-induced respiratory plasticity, such as protein kinase C (Devinney and Mitchell, unpublished), serine/threonine protein phosphatases (Wilkerson et al, 2008) and NADPH oxidase (MacFarlane et al, 2009). …”

“…For example, acute intermittent hypoxia (AIH; 3 hypoxic episodes) elicits a form of respiratory plasticity known as phrenic long-term facilitation (pLTF; Bach and Mitchell, 1996; Matieka and Sandhu, 2011; Mitchell and Terada, 2011; Mitchell et al, 2001). Our understanding of cellular mechanisms underlying AIH-induced pLTF has advanced considerably in recent years (Dale-Nagle et al, 2010; Feldman et al, 2003; MacFarlane et al, 2009; Mahamed and Mitchell, 2007; Mitchell et al, 2001). For example, pLTF requires spinal serotonin type 2 receptor activation (Baker-Herman and Mitchell, 2002; MacFarlane and Mitchell, 2009; MacFarlane et al, 2011), new synthesis of brain derived neurotrophic factor (BDNF; Baker-Herman et al, 2004), activation of the high affinity BDNF receptor, TrkB (Baker-Herman et al, 2004), with subsequent downstream signaling via extracellular regulated MAP kinases (ERK; Hoffman and Mitchell, unpublished; Wilkerson and Mitchell, 2009).…”

Hemodynamic shear stress modulates endothelial cell autophagy: Role of LOX-1

“…Although all these enzymes contribute to oxidative burden, it appears that an initial generation of ROS by NADPH oxidases triggers the release of ROS by other enzyme systems [26,27]. In order to determine the role of ROS in expression of LOX-1 and autophagy, we utilized 2 different inhibitors of NADPH oxidases, DPI and apocynin.…”

Phrenicotomy alters phrenic long-term facilitation following intermittent hypoxia in anesthetized rats

“…The mechanisms underlying LTF have been studied extensively over the last 10 -15 years, primarily in anesthetized animals (reviewed in Refs. 42,49,50). In anesthetized animals, LTF is typically manifest as an increase in the inspiratory burst amplitude of phrenic (19) and/or hypoglossal (XII) extracellular nerve recordings (20).…”