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Nerve growth factor, sphingomyelins, and sensitization in sensory neurons

Grant D. Nicol^*

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

摘要

Because nerve growth factor (NGF) is elevated duringinflammation, plays a causal role in the initiation ofhyperalgesia, and is known to activate the sphingomyelinsignalling pathway, we examined whether NGF and itsputative second messenger, ceramide, could modulate theexcitability of capsaicin-sensitive adult sensory neurons.Using the whole-cell patch-clamp recording technique,exposure of isolated sensory neurons to either 100 ng/mLNGF or 1 mmol/L N-acetyl sphingosine (C2-ceramide)produced a 3-4 fold increase in the number of action potentials(APs) evoked by a ramp of depolarizing current ina time-dependent manner. Intracellular perfusion with bacterialsphingomyelinase (SMase) also increased the numberof APs suggesting that the release of native ceramideenhanced neuronal excitability. Glutathione, an inhibitorof neutral SMase, completely blocked the NGF-inducedaugmentation of AP firing, whereas dithiothreitol, an inhibitorof acidic SMase, was without effect. In the presenceof glutathione and NGF, exogenous ceramide still enhancedthe number of evoked APs, indicating that the sensitizingaction of ceramide was downstream of NGF. Toinvestigate the mechanisms of actions for NGF andceramide, isolated membrane currents were examined.Both NGF and ceramide facilitated the peak amplitude ofthe TTX-resistant sodium current (TTX-R INa) by approximately1.5-fold and shifted the activation to morehyperpolarized voltages. In addition, NGF and ceramidesuppressed an outward potassium current (IK) by ~35%.The inflammatory prostaglandin, PGE2, produced an additionalsuppression of IK after exposure to ceramide(~35%), suggesting that these agents might act on differenttargets.Based on the existing literature, it is not clear whetherthis NGF-induced sensitization is mediated by the highaffinityTrkA receptor or the low-affinity p75 neurotrophinreceptor. Pretreatment with the p75 blocking antibodycompletely prevents the NGF-induced increase in the numberof APs evoked by the current ramp. Although thesensitization by NGF was blocked, the antibody had noeffect on the capacity of ceramide, a putative downstreamsignalling molecule, to enhance the excitability.Ceramide can be metabolized by ceramidase to sphingosine(Sph) and Sph to sphingosine 1-phosphate (S1P)by sphingosine kinase. It is well established that each ofthese products of sphingomyelin metabolism can act asintracellular signalling molecules. This raises the questionas to whether the enhanced excitability produced by NGFwas mediated directly by ceramide or required additionalmetabolism to Sph and/or S1P. Sph applied externally didnot affect the neuronal excitability whereas internally perfusedSph augmented the number of APs evoked by thedepolarizing ramp. Furthermore, internally perfused S1Penhanced the number of evoked APs. This sensitizing actionof NGF, ceramide, and internally perfused Sph, wereabolished by dimethylsphingosine (DMS), an inhibitor ofsphingosine kinase. In contrast, internally perfused S1Penhanced the number of evoked APs in the presence ofDMS. These observations support the idea that the metabolismof ceramide/Sph to S1P is critical for the sphingolipid-induced modulation of excitability. Thus, our findingsindicate that the pro-inflammatory agent, NGF, canrapidly enhance the excitability of sensory neurons. ThisNGF-induced sensitization is mediated by activation of thesphingomyelin signalling pathway wherein intracellular S1Pderived from ceramide, acts as an internal second messengerto regulate membrane excitability, however, the effectorsystem whereby S1P modulates excitability remainsundetermined.

Nerve growth factor, sphingomyelins, and sensitization in sensory neurons

Grant D. Nicol^*

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

Abstract

Because nerve growth factor (NGF) is elevated duringinflammation, plays a causal role in the initiation ofhyperalgesia, and is known to activate the sphingomyelinsignalling pathway, we examined whether NGF and itsputative second messenger, ceramide, could modulate theexcitability of capsaicin-sensitive adult sensory neurons.Using the whole-cell patch-clamp recording technique,exposure of isolated sensory neurons to either 100 ng/mLNGF or 1 mmol/L N-acetyl sphingosine (C2-ceramide)produced a 3-4 fold increase in the number of action potentials(APs) evoked by a ramp of depolarizing current ina time-dependent manner. Intracellular perfusion with bacterialsphingomyelinase (SMase) also increased the numberof APs suggesting that the release of native ceramideenhanced neuronal excitability. Glutathione, an inhibitorof neutral SMase, completely blocked the NGF-inducedaugmentation of AP firing, whereas dithiothreitol, an inhibitorof acidic SMase, was without effect. In the presenceof glutathione and NGF, exogenous ceramide still enhancedthe number of evoked APs, indicating that the sensitizingaction of ceramide was downstream of NGF. Toinvestigate the mechanisms of actions for NGF andceramide, isolated membrane currents were examined.Both NGF and ceramide facilitated the peak amplitude ofthe TTX-resistant sodium current (TTX-R INa) by approximately1.5-fold and shifted the activation to morehyperpolarized voltages. In addition, NGF and ceramidesuppressed an outward potassium current (IK) by ~35%.The inflammatory prostaglandin, PGE2, produced an additionalsuppression of IK after exposure to ceramide(~35%), suggesting that these agents might act on differenttargets.Based on the existing literature, it is not clear whetherthis NGF-induced sensitization is mediated by the highaffinityTrkA receptor or the low-affinity p75 neurotrophinreceptor. Pretreatment with the p75 blocking antibodycompletely prevents the NGF-induced increase in the numberof APs evoked by the current ramp. Although thesensitization by NGF was blocked, the antibody had noeffect on the capacity of ceramide, a putative downstreamsignalling molecule, to enhance the excitability.Ceramide can be metabolized by ceramidase to sphingosine(Sph) and Sph to sphingosine 1-phosphate (S1P)by sphingosine kinase. It is well established that each ofthese products of sphingomyelin metabolism can act asintracellular signalling molecules. This raises the questionas to whether the enhanced excitability produced by NGFwas mediated directly by ceramide or required additionalmetabolism to Sph and/or S1P. Sph applied externally didnot affect the neuronal excitability whereas internally perfusedSph augmented the number of APs evoked by thedepolarizing ramp. Furthermore, internally perfused S1Penhanced the number of evoked APs. This sensitizing actionof NGF, ceramide, and internally perfused Sph, wereabolished by dimethylsphingosine (DMS), an inhibitor ofsphingosine kinase. In contrast, internally perfused S1Penhanced the number of evoked APs in the presence ofDMS. These observations support the idea that the metabolismof ceramide/Sph to S1P is critical for the sphingolipid-induced modulation of excitability. Thus, our findingsindicate that the pro-inflammatory agent, NGF, canrapidly enhance the excitability of sensory neurons. ThisNGF-induced sensitization is mediated by activation of thesphingomyelin signalling pathway wherein intracellular S1Pderived from ceramide, acts as an internal second messengerto regulate membrane excitability, however, the effectorsystem whereby S1P modulates excitability remainsundetermined.

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通讯作者：Grant D. Nicol　　E-mail: Grant D. Nicol

引用本文：

Grant D. Nicol. Nerve growth factor, sphingomyelins, and sensitization in sensory neurons[J]. 生理学报 2008; 60 (5): 603-604.

Grant D. Nicol. Nerve growth factor, sphingomyelins, and sensitization in sensory neurons. Acta Physiol Sin 2008; 60 (5): 603-604 (in Chinese with English abstract).