Spinal 5-HT7 receptors and protein kinase A constrain intermittent hypoxia-induced phrenic long-term facilitation

Abstract

Phrenic long-term facilitation (pLTF) is a form of serotonin-dependent respiratory plasticity induced by acute intermittent hypoxia (AIH). pLTF requires spinal Gq protein-coupled serotonin-2 receptor (5-HT2) activation, new synthesis of brain-derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, TrkB. Intrathecal injections of selective agonists for Gs protein-coupled receptors (adenosine 2A and serotonin-7; 5-HT7) also induce long-lasting phrenic motor facilitation via TrkB “trans-activation.” Since serotonin release near phrenic motor neurons may activate multiple serotonin receptor subtypes, we tested the hypothesis that 5-HT7 receptor activation contributes to AIH-induced pLTF. A selective 5-HT7 receptor antagonist (SB-269970, 5mM, 12μl) was administered intrathecally at C4 to anesthetized, vagotomized and ventilated rats prior to AIH (3, 5-min episodes, 11% O2). Contrary to predictions, pLTF was greater in SB-269970 treated versus control rats (80±11% vs 45±6% 60 min post-AIH; p<0.05). Hypoglossal LTF was unaffected by spinal 5-HT7 receptor inhibition, suggesting that drug effects were localized to the spinal cord. Since 5-HT7 receptors are coupled to protein kinase A (PKA), we tested the hypothesis that PKA inhibits AIH-induced pLTF. Similar to 5-HT7 receptor inhibition, spinal PKA inhibition (KT-5720, 100μM, 15μl) enhanced pLTF (99±15% 60 min post-AIH; p<0.05). Conversely, PKA activation (8-br-cAMP, 100μM, 15μl) blunted pLTF versus control rats (16±5% vs 45±6% 60 min post-AIH; p<0.05). These findings suggest a novel mechanism whereby spinal Gs protein-coupled 5-HT7 receptors constrain AIH-induced pLTF via PKA activity.

Spinal 5-HT7 receptors induce phrenic motor facilitation via EPAC-mTORC1 signaling

“…To determine whether PKA is necessary for 5-HT 7 receptor-induced pMF KT-5720, a PKAi (n ϭ 6; 12 l, 100 M), was given prior to 5-HT 7 receptor agonist injections. We previously demonstrated that KT-5720 at this dose prevents PKA-mediated constraint of 5-HT 2A receptor-dependent, AIH-induced pMF (Hoffman and Mitchell 2013). In addition, this dose prevents PKA-but not EPAC-dependent signaling within cell cultures (Davies et al 2000).…”

“…Although PKA constrains 5-HT 2 receptordependent pMF through cross-talk inhibition (Hoffman and Mitchell 2013), several lines of evidence suggest that EPAC (not PKA) mediates 5-HT 7 receptor-induced pMF. However, no study has attempted to localize these proteins in spinal respiratory neurons in general, or phrenic motor neurons (PMNs) in specific.…”

“…2, A and Biv). The KT-5720 dose is based on previous work using the same in vivo protocol (Hoffman and Mitchell 2013) and PKA cell culture activity assays (Davies et al 2000). Cervical spinal injections of the 5-HT 7 receptor agonist did not affect hypoglossal nerve (XII) activity (n ϭ 4; 90 min: Ϫ0.1 Ϯ 13.1%, P ϭ 0.95), suggesting that 5-HT 7 receptor agonistinduced pMF requires activation of spinal (vs. brain stem) receptors (Baker-Herman and Mitchell 2002;MacFarlane and Mitchell 2009).…”

“…While the S and Q pathways elicit pMF through distinct signaling cascades, they interact via mutual "cross-talk inhibition" (Dale-Nagle et al 2010;Devinney et al 2013;Hoffman and Mitchell 2013). One manifestation of cross-talk inhibition is the bell-shaped dose-response curve of pMF in response to intermittent intrathecal serotonin injections (MacFarlane and Mitchell 2009).…”

“…Thus, when activated alone, 5-HT 2 (MacFarlane et al 2011) and 5-HT 7 (Hoffman and Mitchell 2011) receptors each give rise to distinct forms of pMF. When activated concurrently, they effectively cancel each other because of balanced cross-talk inhibition (Devinney et al 2013;Hoffman and Mitchell 2013;MacFarlane and Mitchell 2009). Cross-talk inhibition from 5-HT 7 receptors to Q pathway-induced pMF is mediated by protein kinase A (PKA) activity (Hoffman and Mitchell 2013); however, it is not known how 5-HT 7 receptor activation initiates pMF when acting alone (Hoffman and Mitchell 2011).…”