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Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin-sensitive adult and embryonic sensory neurons. Using the whole-cell patch-clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml−1 NGF or 1 μM N-acetyl sphingosine (C2-ceramide) produced a 3- to 4-fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time-dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF-induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX-resistant sodium current (TTX-R INa) by approximately 1.5-fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (IK) by ≈35 %. Ceramide reduced IK in a concentration-dependent manner. Isolation of the NGF- and ceramide-sensitive currents indicates that they were delayed rectifier types of IK. The inflammatory prostaglandin, PGE2, produced an additional suppression of IK after exposure to ceramide (≈35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro-inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF-induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.
Ceramides are novel second messengers that may mediate the inflammatory response, apoptosis and altered gene expression in different cell types. One component of the inflammatory response involves activation of small diameter sensory neurons which, in turn, contributes to heightened sensitivity, vasodilatation and plasma extravasation. Indeed, a number of inflammatory mediators including prostaglandins (Kress & Reeh, 1996), NGF (Lewin et al. 1993; Lewin & Mendell, 1993; Shu & Mendell, 1999a), and cytokines (Ferreira et al. 1988; Schweizer et al. 1988; Cunha et al. 1992) enhance the sensitivity of sensory neurons to noxious stimulation. Although the prostaglandin-induced sensitization results from activation of the cyclic AMP- protein kinase A (PKA) transduction cascade (Cui & Nicol, 1995; Hingtgen et al. 1995; Evans et al. 1999) the second messenger system(s) that mediate the effects of these neurotrophins and cytokines remains unknown. This raises the question of whether NGF and/or ceramide plays any role in this enhanced sensitivity of sensory neurons.
In a number of cell systems, activation of cytokine receptors results in the liberation of ceramides as second messenger signalling molecules (Schütze et al. 1994; Ballou et al. 1996). In addition, recent studies have shown that NGF binding to the low-affinity neurotrophin receptor (p75NTR) activates the sphingomyelin signalling pathway causing the liberation of ceramide (Dobrowsky et al. 1994). Ceramide is thought to act as a second messenger molecule capable of mediating multiple physiological effects and this depends on the cell type in question (Schütze et al. 1994; Ballou et al. 1996; Mathias et al. 1998). Such effects range from the regulation of cell growth and apoptosis to the immune response. Based on the notion that certain pro-inflammatory agents, such as NGF, might activate the sphingomyelin signalling pathway, we explored the idea that ceramide, acting as an intracellular messenger, alters the sensitivity of sensory neurons to excitatory stimulation. In this report we demonstrate that NGF, through a pathway that depends on activation of a neutral sphingomyelinase (SMase), and ceramide enhance the excitability of sensory neurons through the modulation of both a TTX-R INa and a voltage-dependent IK. Portions of this work have been published previously in abstract form (Zhang et al. 2000; Nicol et al. 2001).
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In this report, we demonstrate that NGF and ceramide directly enhance the capacity of capsaicin-sensitive sensory neurons to generate APs. NGF and ceramide increased the rate at which the ramp depolarized the neuron and could thus account for the decreased latency of firing. Although the number of evoked APs was increased after treatment, the apparent threshold for AP firing was not changed. These results suggest that NGF and ceramide do not alter the membrane properties that set the firing voltage but rather those properties that determine how rapidly it achieves that voltage. The focus of future studies will examine how the modulation of TTX-R INa and IK (and perhaps other currents) is integrated to alter the firing of the AP and thus the coding properties of these NGF-sensitive sensory neurons. NGF and ceramide modify a TTX-R INa and IK in both embryonic and adult sensory neurons in a manner that is consistent with the characteristics of the augmented neuronal excitability. Because similar effects were observed in both adult and embryonic neurons this demonstrates that the embryonic neurons can be a useful model system in which to study the effects of inflammatory agents on neuronal excitability.
In sensory neurons and other cell types, long-term exposure to NGF can increase the current density or expression levels of sodium channels (Mandel et al. 1988; Omri & Meiri, 1990; Aguayo & White, 1992; Fjell et al. 1999). To date, there is little if any information on whether acute administration of NGF affects sodium channels. Our observations indicate that both NGF and ceramide rapidly enhanced the TTX-R INa by ≈1.3-fold, but this may be lower than actual values because a prepulse to remove inactivation was not used, thus some portion of TTX-R INa may have been inactivated. Also, NGF or ceramide shifted activation of TTX-R INa to more hyperpolarized voltages. We believe that this shift did not result from a loss of space clamp in these neurons because the current-voltage relation remained graded with depolarization. Both the slope factor, k, and the reversal potential for TTX-R INa (calculated to be +28 mV) did not change over the entire recording period. Furthermore, after a 20 min exposure to normal recording solution or dihydroceramide the peak TTX-R INa did not shift to more hyperpolarized voltages nor were G/Gmax or k altered. These findings suggest that the clamp was maintained during the recording period and that this current was stable throughout our period of recording.
Recent work by Rush et al. (1998) suggests that in adult rat sensory neurons there might be three different types of TTX-R INa. Molecular studies originally described the PN3/SNS subtype (Akopian et al. 1996; Sangameswaran et al. 1996) and more recent studies have found another distinct TTX-R subtype known as SNS2 (Tate et al. 1998) or NaN (Dib-Hajj et al. 1998b). The mRNA levels for SNS and NaN decreased after axotomy with corresponding changes in the currents (Dib-Hajj et al. 1996, 1998b; Cummins & Waxman, 1997; but see Novakovic et al. 1998). The decreased expression of SNS after axotomy could be reversed by chronic treatment (10-12 days) with NGF (Dib-Hajj et al. 1998a) suggesting that NGF plays an important role in the expression of SNS. Interestingly, inflammation produced by complete Freund's adjuvant increased the expression levels of either PN3 (see Porreca et al. 1999) or SNS2 (Tate et al. 1998) rather than decreasing them as observed with the axotomy model. The physiological significance as to why injury causes a decrease whereas inflammation causes an increase in TTX-R sodium channel expression remains to be determined. Because these studies examined sodium channel expression over long periods of treatment, i.e. days, rather than the short time periods as in our study, it is difficult to speculate upon which TTX-R subtypes NGF/ceramide might act.
We found that NGF and ceramide suppressed an outward IK that was probably a delayed rectifier type of current based on its activation voltage, rapid activation, and slow relaxation kinetics. Although both agents reduced IK, it remains to be determined why ceramide shifted the half-activation voltage to more hyperpolarized potentials (≈10 mV) whereas NGF had no significant effect (Table 3). One possibility is that NGF may have other parallel effects that are not dependent on the actions of ceramide. This notion is, however, not consistent with the observation that GSH completely suppressed the capacity of NGF to augment the AP firing. This is a question that must be resolved in future studies examining the actions of NGF on IK. Unlike the sodium current, more is known about the actions of ceramide on IK. Ceramide inhibited a calcium-dependent IK in smooth muscle cells from coronary artery (Li et al. 1999). In T lymphocytes, ceramide inhibited the Kv1.3-type potassium channel through activation of a Src-like tyrosine kinase (Gulbins et al. 1997) and in oligodendrocytes ceramide blocks an inward rectifier type IK by a ras and raf-1 pathway (Hida et al. 1998). Thus, ceramide appears to act on a variety of potassium currents in a number of cell types through different pathways, and thus modulation of different potassium channels could give rise to enhanced neuronal excitability.
In behavioural models of nociception, treatment with NGF increases the sensitivity to both noxious mechanical and thermal stimulation (Lewin et al. 1993; Woolf et al. 1994; Rueff & Mendell, 1996; Shu & Mendell, 1999a). The time course for sensitization of the thermal response was much shorter (tens of minutes) than the mechanical response (hours). Like the in vivo thermal response, sensitization of isolated sensory neurons by NGF was rapid, reaching maximal effects in ≈6 min. Because NGF acted rapidly on capsaicin-sensitive neurons, we would predict that the neurons we recorded from might correspond, in part, to those neurons whose thermal sensitivity was enhanced by NGF (Lewin et al. 1993). Thus, our findings in isolated sensory neurons are consistent with those made in in vivo models and additionally show that NGF acts directly on sensory neurons rather than through some other immunocompetent cell type to alter neuronal sensitivity.
Based on our finding that the NGF-induced enhancement of excitability was blocked by inhibition of SMase, we speculate that NGF could be acting via p75NTR. This notion is consistent with earlier work where NGF elevated ceramide levels through activation of p75NTR in T9 glioma cells (Dobrowsky et al. 1994). Based on this notion and the observation that p75NTR is co-expressed in nearly all of trk-expressing sensory neurons (≈76 % of the total DRG neurons express trk mRNA; Wright & Snider, 1995) our results would predict that NGF should produce thermal or mechanical hyperalgesia, in part, by sensitizing sensory neurons. Recent results indicated that NGF's capacity to elicit hyperalgesia was through activation of TrkANTR only because injection of NGF in p75NTR knockout mice (Lee et al. 1992) produced both mechanical and thermal hyperalgesia (Bergmann et al. 1998). However, these results must be interpreted with caution in the light of new work. von Schack et al. (2001) showed that the p75NTR‘knockout’ generated by Lee et al. (1992) removed only three of the four cysteine-rich binding domains, which may permit expression of a functional protein. Deletion of all four domains resulted in reduced numbers of Schwann cells as well as a greater loss of neurons in L5 DRG compared to the three of four domain deletion (54 % decrease vs. 39 %, respectively). It will be quite interesting to determine if NGF produces hyperalgesia in the four of four domain deletion p75 knockout mice.
Shu & Mendell (1999b) have shown that a 10 min exposure to 100 ng ml−1 of NGF or NT-4/5 augmented by about two-fold the capsaicin-evoked current (ICAP) in rat sensory neurons. This is thought to result from activation of either TrkANTR or TrkBNTR because the enhancement was blocked by K-252a, a presumed selective inhibitor of TrkNTR tyrosine kinase activity. NT-3 had no effect on ICAP. If the actions of NGF were mediated entirely by the p75NTR, then one might expect NT-3 to augment ICAP. The reasons for the lack of effect by NT-3 are not apparent. The role of NT-3 in sensitization of sensory neurons is complicated by the observation that NT-3 suppressed the electrically evoked release of substance P in an isolated spinal cord preparation (Malcangio et al. 1997). These results suggest that NT-3 might be anti-nociceptive. Recently, Shu & Mendell (2001) showed that the NGF-induced enhancement of ICAP was suppressed partially by inhibitors of PKA. Interestingly, K-252a, the tyrosine kinase inhibitor, can block other kinases like PKA and PKC (Kase et al. 1987). Because of the lack of selectivity of K-252a, it is difficult to conclude that NGF sensitizes ICAP solely through activation of the TrkANTR. Thus, NGF may sensitize sensory neurons through multiple signalling pathways whose interactions are, at present, poorly understood.
The observation that NGF acutely enhanced the excitability of these sensory neurons, which have been grown in the presence of NGF, raises interesting questions regarding the apparent lack of desensitization of this particular receptor/pathway. NGF remaining from the culture medium was washed away with superfusion of normal Ringer solution so that these neurons were free of NGF for approximately 30 min prior to re-exposure. Our results suggest that this NGF-free period is sufficient to establish suitable conditions for NGF induction of sensitization (if such a process is necessary). Consistent with this notion is the finding that the half-time for dissociation of NGF from its low-affinity binding site (site II) is approximately 3 s (Sutter et al. 1979). The issue of desensitization is complicated by our finding that after a 10-30 min washout, neither the ceramide-induced enhancement of AP firing nor the NGF-induced suppression of IK was diminished. In contrast, the NGF-induced enhancement of ICAP returned to control values ≈10 min after removal of NGF, although if external calcium was removed, the enhancement by NGF did not recover (> 1 h; Shu & Mendell, 2001). Desensitization of ICAP can be prevented by the removal of external calcium (Cholewinski et al. 1993; Docherty et al. 1996; Koplas et al. 1997). Therefore, it is difficult to ascertain directly whether recovery of the NGF-induced sensitization of ICAP in normal calcium was due to a process specific to the NGF pathway or whether the recovery was part of the tachyphylaxis of the capsaicin-gated ion channel. It is possible that reversal of the sensitization of the capsaicin-gated current and the sodium and potassium currents exhibit different time courses due to differences in signalling pathways. Additional studies are required to understand the pathways activated by NGF and how these pathways modulate the activity of different ion channels.
Our findings raise an interesting question as to the effector system(s) whereby ceramide modulates the activity of neuronal ion channels. Ceramide can stimulate directly a ceramide-activated protein kinase (CAPK, a serine/ threonine type; Joseph et al. 1993; Mathias et al. 1993; Liu et al. 1994) and a ceramide-activated protein phosphatase (CAPP, a member of the serine/threonine type 2A group; Dobrowsky & Hannun, 1992; Dobrowsky et al. 1993). It seems unlikely that ceramide sensitizes sensory neurons through activation of a phosphatase. Okadaic acid, an inhibitor of phosphatases 1 and 2A, augmented the evoked-release of substance P and calcitonin gene-related peptide from sensory neurons (Hingtgen & Vasko, 1994). Facilitation of peptide release is not consistent with a ceramide-induced increase in phosphatase activity producing the increased AP firing in sensory neurons. Although the roles of CAPK and CAPP in regulating neuronal excitability have not been explored, our observations suggest that modulation of these currents results from kinase(s), rather than phosphatase, activity.
The inflammatory prostaglandins, notably PGI2 and PGE2, produce hyperalgesia in behavioural models (Kress & Reeh, 1996) and also enhance the excitability of sensory neurons (Hingtgen et al. 1995; Nicol et al. 1997). Interestingly, both the TTX-R INa and IK currents are modulated by ceramide in a manner similar to that produced by PGE2. We have shown previously that PGE2 increased the number of evoked APs via cyclic AMP-dependent activation of protein kinase A (PKA; Cui & Nicol, 1995; Nicol et al. 1997). In sensory neurons, PKA plays a critical role in augmenting the amplitude and rate of inactivation of the TTX-R INa (England et al. 1996; Gold et al. 1996). In addition, activation of PKA results in a suppression of a delayed rectifier-like IK (Evans et al. 1999). Therefore, modulation of these currents could result from activation of multiple protein kinases. In support of this notion, we found that PGE2 could further suppress IK by ≈35 % after exposure to ceramide. This suggests that these agents act on different channel subtypes or that their different effector actions may be additive at the same target. During inflammation it is possible that there are multiple parallel signalling pathways that either together or separately converge on the TTX-R INa and IK currents. Therefore, modulation of these two currents, and possibly others, by inflammatory mediators can result in the heightened sensitivity to noxious stimulation observed in behavioural models. Future studies exploring the roles of the sphingomyelin signalling pathway may prove significant in our understanding of how neurotrophins and novel signalling lipids contribute to the regulation of neuronal activity.