- Email: [email protected]
Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells
Neuroscience Letters 248 (1998) 77–80
Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells Sandra Lourenssen a, Michael G. Blennerhassett b ,* a
Department of Fetal Health and Development, Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario MSG 1X5, Canada b Department of Pathology, McMaster University Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada
Received 20 January 1998; received in revised form 23 March 1998; accepted 23 March 1998
Abstract Since lysophosphatidylserine (LPS) is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells, we investigated whether LPS might potentiate the effects of NGF in inducing neural differentiation of PC12 cells. Cell morphology was evaluated 48 h after addition of NGF, LPS or NGF + LPS. LPS alone was ineffective, but strongly promoted NGF-induced differentiation to give rise to cells that more closely resembled neurons in primary culture. LPS increased the number of PC12 cells that developed neurites in response to NGF (0.01–40 ng/ml), with the response to 1.0 ng/ml increasing from 17.8 ± 2.2 to 50.8 ± 4.1% when LPS was also present. Neurite length was also greater in PC12 cells receiving NGF + LPS: 17.8 ± 2.2% of cells had neurites longer than three cell body diameters with 1.0 ng/ml NGF + 1 mg/ml LPS, compared to 1.6 ± 1.6% with NGF alone. Further, cells responding to NGF + LPS typically developed only 1–2 neurites per cell (90.9%, 1 mg/ml LPS), compared with the multipolar appearance with NGF alone (71.1% with 3–6 neurites, 10 ng/ml NGF). LPS occurs at sites of tissue damage where NGF can also be present, and therefore may be a naturally-occurring modifier of neuronal structure and/or function. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Lysophosphatidylserine; Nerve growth factor; Neurites; PC12 cells; Tissue culture
The neurotrophin nerve growth factor (NGF) has many varied actions on both neuronal and non-neuronal cells, mediated by binding of this neurotrophic factor to two classes of receptors expressed on the surface of responsive cells [11]. NGF binds with high-affinity to the tyrosine kinase receptor trkA and activation of trkA is thought to be essential for NGF function [6]. NGF also binds to a low-affinity receptor, p75NGFR, and functional assays have indicated that p75NGFR can cooperate with trk receptors to increase the affinity of neurotrophin binding and/or the signalling efficiency [2]. An important non-neuronal function of NGF is its highly potent ability to induce secretion of inflammatory mediators such as histamine and serotonin from rat peritoneal mast cells [10]. However, this requires the presence of lysopho* Corresponding author. Tel.: +1 905 5259140 ext. 22771; fax: +1 905 5212613; e-mail: [email protected]
sphatidylserine (LPS) [4,13]. LPS is thought to be generated by acylation of phosphatidylserine upon tissue damage, and may be a soluble messenger of tissue injury [5]. Thus, NGF can activate the trkA signalling pathway, but requires modulation of at least one component of this signal transduction cascade by LPS to cause mast cell activation. Since mast cells have been shown to express the trkA receptor, but not p75NGFR [10], LPS may serve a collaborative function similar to that of the low affinity receptor elsewhere. We tested whether LPS would also interact with the trkA signalling pathway in a neural environment, using the PC12 cell line that expresses both trkA and p75NGFR [6]. PC12 cells respond to NGF by growth arrest and differentiation into a neuronal phenotype with multiple neurites [9]. We have analyzed the effect of LPS on NGF-induced morphological differentiation of PC12 cells. PC12 cells were maintained as described [9], and 24 h prior to NGF addition, 2 × 104 cells were added to each well
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00275- 4
78
S. Lourenssen, M.G. Blennerhassett / Neuroscience Letters 248 (1998) 77–80
Fig. 1. Phase contrast micrographs showing LPS potentiation of NGF-induced neuronal phenotype in PC12 cells. Cells were exposed 48 h earlier to control medium (a), 1 ng/ml NGF (b) or 1 ng/ml NGF + 10 mg/ml LPS (c). Arrow, growth cone. Scale bar, 40 mm.
of a collagen-coated 24-well plate. On the following day, the media was changed, and fresh media containing varying concentrations of NGF and/or LPS were added to the wells in triplicate. After 48 h, the media was removed and the cells were fixed in 10% formalin. At least 100 cells were analysed for the length and number of neurites in a total of at least three wells per condition, and all experiments were repeated four times. The addition of NGF alone to PC12 cells resulted in major changes in their appearance at 48 h (Fig. 1a,b), suggestive of differentiation into a neuronal phenotype as described previously [9]. NGF caused a dose-dependent increase in cells with neurites longer than one or three cell body diameters (Fig. 2). With 0.01 ng/ml NGF, very few cells extended neurites greater than one cell diameter, while NGF at ≥10 ng/ml caused a maximal response, with .70% having neurites greater than one cell body diameter. While LPS alone had no effect on morphology, LPS added concurrently with NGF had a dramatic effect on the appearance of PC12 cells (Fig. 1). PC12 cells stimulated with 1 ng/ml NGF + 10 mg/ml LPS developed far fewer neurites per cell, typically forming a single very long neurite (Fig. 1c). Furthermore, the cell bodies appeared phasebright with a more rounded appearance (Fig. 2b,c), and the neurites were thinner, with a well-defined growth cone (Fig. 2c), suggesting an altered interaction with the culture surface. Overall, PC12 cells receiving NGF + LPS were more similar to primary neurons in culture than cells receiving NGF alone. The presence of LPS also strongly potentiated the number of PC12 cells responding to NGF, as well as the magnitude of their response (Fig. 3). The number of PC12 cells responding to 1 ng/ml NGF with neurites greater than one cell body diameter more than doubled when LPS (1 mg/ml) was also present, increasing from 20.8 ± 4.0 to 42.2 ± 6.1%. After exposure to 1 ng/ml NGF, the number of PC12 cells showing neurites greater than three cell body diameters in length was routinely low, at 1.0 ± 1.6%, but increased prominently to 15.7 ± 3.8%when 1 mg/ml LPS was also pre-
sent. When LPS was increased to 10 mg/ml, no significant further increase in effect was observed. Addition of a monoclonal antibody to 2.5S NGF prevented morphological differentiation by NGF either alone or in combination with LPS (data not shown). PC12 cells that responded to NGF by the development of neurites generally showed a multipolar appearance. While cells with one to six neurites were observed, about 80% of the responding cells had two to four neurites/cell, and this was not affected by the concentration of NGF over a range of 0.1–10 ng/ml (Fig. 4a). However, PC12 cells exposed to NGF in the presence of LPS tended to be unipolar or bipolar, as seen in Fig. 1c, and this was confirmed by examination of the distribution of the number of neurites per responding cell (i.e. at least one neurite; Fig. 4). For example, in the presence of 0.1 ng/ml NGF, 34.6% of responding
Fig. 2. Dose-dependency of NGF-induced outgrowth of neurites from PC12 cells, showing presence of neurites at least one or three cell body diameters (CBD) in length. Data are the mean ± SEM.
S. Lourenssen, M.G. Blennerhassett / Neuroscience Letters 248 (1998) 77–80
79
signalling mechanism is also activated in PC12 cells following the addition of NGF [7], an enhanced turnover of phospholipids may be involved in the morphological changes in the PC12 cells that occur upon the addition of NGF and LPS. LPS may be generated after damage to the cell membrane, and therefore could be involved with the action of NGF in the regulation of inflammatory and immune reactions through its effects on mast cells [5]. Our study has shown that LPS can also strongly potentiate the action of NGF as a neurotrophin. Since sympathetic neurons remain dependent on NGF in the adult [14], LPS may be a naturally occurring factor affecting neural regeneration in the periphery. Further, this interaction may have important functional consequences on neurons at sites of damage or inflammation, through sensitization of nociceptors. Recent evidence has implicated NGF as the major factor mediating inflammatory hyperalgesia, where LPS may act both indirectly through the activation of mast cells or post-ganglionic sympathetic fibres [1,12] and directly through the sensitization Fig. 3. LPS increases the response of PC12 cells to NGF. LPS added to NGF (1 ng/ml) increased the percent of cells with neurites longer than one or three cell body diameters (CBD) compared with NGF alone. Data are the mean ± SEM; *P , 0.05 relative to control.
PC12 cells had three neurites, while 13.5% extended a single neurite (Fig. 4a). Addition of LPS (1 mg/ml) changed this significantly: only 6.5% of cells had three neurites, and 55.8% of cells had one neurite (Fig. 4b). While 24.1% of cells exposed to 1 ng/ml NGF had five neurites, only 6.3% of cells had five neurites when LPS was also present. No cells with more than four neurites were detected under those conditions. To determine whether LPS would maintain the differentiated state, the media was changed 48 h after addition of NGF and LPS, and media alone or containing LPS without NGF was added. After 24 h, significant retraction of neurites had occurred, which was not affected by the presence of LPS (data not shown). Therefore, the more strongly differentiated state that results from the addition of NGF + LPS requires the presence of NGF for its maintenance. The effects of compounds related to LPS were also tested. Neither lysophosphatidylcholine nor lysophosphatidylinositol affected the morphology of PC12 cells, indicating the absolute requirement for the serine group. Sphingomyelin was also tested, since NGF-induced sphingomyelin metabolism leads to the production of ceramide, implicated in growth arrest and differentiation [8], but no effect was seen. Therefore, the effects of LPS seem specific, and are not a general outcome of the presence of a lipid molecule. Our results provide evidence that LPS can modulate the effects of NGF in a neuronal cell type. While it is possible that LPS acts at the cell surface to facilitate the interaction between p75 and trkA, addition of LPS and NGF to mast cells led to an increased activation of phospholipase C and phosphoinositide hydrolysis [3]. Since the phosphoinositide
Fig. 4. Distribution of neurite number among PC12 cells responding to either NGF alone (A) or NGF + LPS (B). Scale bars, mean ± SEM; *P , 0.05 relative to NGF alone.
80
S. Lourenssen, M.G. Blennerhassett / Neuroscience Letters 248 (1998) 77–80
of sensory neurons. Further studies in vivo may elucidate a potential role of LPS in neuronal sensitization after injury. [1] Andreev, N.Y., Dimitrieva, N., Koltzenburg, M. and McMahon, S.B., Peripheral administration of NGF in the adult rat produces a thermal hyperalgesia that requires the presence of sympathetic post-ganglionic neurons, Pain, 63 (1995) 109–115. [2] Barker, P.A. and Shooter, E.M., Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to trkA on PC12 cells, Neuron, 13 (1994) 203–215. [3] Bellini, F., Toffano, G. and Bruni, A., Activation of phosphoinositide hydrolysis by nerve growth factor and lysophosphatidylserine in rat peritoneal mast cells, Biochim. Biophys. Acta, 970 (1988) 187–193. [4] Bruni, A., Bigon, E., Boarato, E., Mietto, L., Leon, A. and Toffano, G., Interaction between nerve growth factor and lysophosphatidylserine on rat peritoneal mast cells, FEBS Lett., 138 (1982) 190–192. [5] Bruni, A., Monastra, G., Bellini, F. and Toffano, G., Autocoid properties of lysophosphatidylserine, Prog. Clin. Biol. Res., 282 (1988) 165–179. [6] Chao, M.V., Neurotrophin receptors: a window into neuronal differentiation, Neuron, 9 (1992) 583–593. [7] Contreras, M.L. and Guroff, G., Calcium-dependent nerve
[8]
[9]
[10]
[11] [12]
[13]
[14]
growth factor-stimulated hydrolysis of phosphoinositides inPC12 cells, J. Neurochem., 48 (1987) 1466–1472. Dobrowsky, R.T., Jenkins, G.M. and Hannun, Y.A., Neurotrophins induce sphingomyelin hydrolysis, J. Biol. Chem., 270 (1995) 22135–22142. Greene, L.A., Aletta, J.M., Rukenstein, A. and Green, S.H., PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation, Methods Enzymol., 147 (1986) 207–216. Horigome, K., Pryor, J.C., Bullock, E.D. and Johnson, E.M., Mediator release from mast dells by nerve growth factor. Neurotrophin specificity and receptor mediation, J. Biol. Chem., 268 (1993) 14881–14887. Levi-Montalcini, R., The nerve growth factor: thirty five years later, EMBO J., 6 (1987) 1145–1154. Lewin, G.R., Rueff, A. and Mendell, L.M., Peripheral and central mechanisms of NGF-induced hyperalgesia, Eur. J. Neurosci., 6 (1994) 1903–1912. Mazurek, N., Weskamp, G., Erne, P. and Otten, U., Nerve growth factor induces mast cell degranulation without changing intracellular calcium levels, FEBS Lett., 198 (1986) 315–320. Ruit, K.G., Osborne, P.A., Schmidt, R.E., Johnson, E.M. Jr. and Snider, W.D., Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse, J. Neurosci., 10 (1990) 2412–2419.
Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells Sandra Lourenssen a, Michael G. Blennerhassett b ,* a
Department of Fetal Health and Development, Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario MSG 1X5, Canada b Department of Pathology, McMaster University Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada
Received 20 January 1998; received in revised form 23 March 1998; accepted 23 March 1998
Abstract Since lysophosphatidylserine (LPS) is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells, we investigated whether LPS might potentiate the effects of NGF in inducing neural differentiation of PC12 cells. Cell morphology was evaluated 48 h after addition of NGF, LPS or NGF + LPS. LPS alone was ineffective, but strongly promoted NGF-induced differentiation to give rise to cells that more closely resembled neurons in primary culture. LPS increased the number of PC12 cells that developed neurites in response to NGF (0.01–40 ng/ml), with the response to 1.0 ng/ml increasing from 17.8 ± 2.2 to 50.8 ± 4.1% when LPS was also present. Neurite length was also greater in PC12 cells receiving NGF + LPS: 17.8 ± 2.2% of cells had neurites longer than three cell body diameters with 1.0 ng/ml NGF + 1 mg/ml LPS, compared to 1.6 ± 1.6% with NGF alone. Further, cells responding to NGF + LPS typically developed only 1–2 neurites per cell (90.9%, 1 mg/ml LPS), compared with the multipolar appearance with NGF alone (71.1% with 3–6 neurites, 10 ng/ml NGF). LPS occurs at sites of tissue damage where NGF can also be present, and therefore may be a naturally-occurring modifier of neuronal structure and/or function. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Lysophosphatidylserine; Nerve growth factor; Neurites; PC12 cells; Tissue culture
The neurotrophin nerve growth factor (NGF) has many varied actions on both neuronal and non-neuronal cells, mediated by binding of this neurotrophic factor to two classes of receptors expressed on the surface of responsive cells [11]. NGF binds with high-affinity to the tyrosine kinase receptor trkA and activation of trkA is thought to be essential for NGF function [6]. NGF also binds to a low-affinity receptor, p75NGFR, and functional assays have indicated that p75NGFR can cooperate with trk receptors to increase the affinity of neurotrophin binding and/or the signalling efficiency [2]. An important non-neuronal function of NGF is its highly potent ability to induce secretion of inflammatory mediators such as histamine and serotonin from rat peritoneal mast cells [10]. However, this requires the presence of lysopho* Corresponding author. Tel.: +1 905 5259140 ext. 22771; fax: +1 905 5212613; e-mail: [email protected]
sphatidylserine (LPS) [4,13]. LPS is thought to be generated by acylation of phosphatidylserine upon tissue damage, and may be a soluble messenger of tissue injury [5]. Thus, NGF can activate the trkA signalling pathway, but requires modulation of at least one component of this signal transduction cascade by LPS to cause mast cell activation. Since mast cells have been shown to express the trkA receptor, but not p75NGFR [10], LPS may serve a collaborative function similar to that of the low affinity receptor elsewhere. We tested whether LPS would also interact with the trkA signalling pathway in a neural environment, using the PC12 cell line that expresses both trkA and p75NGFR [6]. PC12 cells respond to NGF by growth arrest and differentiation into a neuronal phenotype with multiple neurites [9]. We have analyzed the effect of LPS on NGF-induced morphological differentiation of PC12 cells. PC12 cells were maintained as described [9], and 24 h prior to NGF addition, 2 × 104 cells were added to each well
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00275- 4
78
S. Lourenssen, M.G. Blennerhassett / Neuroscience Letters 248 (1998) 77–80
Fig. 1. Phase contrast micrographs showing LPS potentiation of NGF-induced neuronal phenotype in PC12 cells. Cells were exposed 48 h earlier to control medium (a), 1 ng/ml NGF (b) or 1 ng/ml NGF + 10 mg/ml LPS (c). Arrow, growth cone. Scale bar, 40 mm.
of a collagen-coated 24-well plate. On the following day, the media was changed, and fresh media containing varying concentrations of NGF and/or LPS were added to the wells in triplicate. After 48 h, the media was removed and the cells were fixed in 10% formalin. At least 100 cells were analysed for the length and number of neurites in a total of at least three wells per condition, and all experiments were repeated four times. The addition of NGF alone to PC12 cells resulted in major changes in their appearance at 48 h (Fig. 1a,b), suggestive of differentiation into a neuronal phenotype as described previously [9]. NGF caused a dose-dependent increase in cells with neurites longer than one or three cell body diameters (Fig. 2). With 0.01 ng/ml NGF, very few cells extended neurites greater than one cell diameter, while NGF at ≥10 ng/ml caused a maximal response, with .70% having neurites greater than one cell body diameter. While LPS alone had no effect on morphology, LPS added concurrently with NGF had a dramatic effect on the appearance of PC12 cells (Fig. 1). PC12 cells stimulated with 1 ng/ml NGF + 10 mg/ml LPS developed far fewer neurites per cell, typically forming a single very long neurite (Fig. 1c). Furthermore, the cell bodies appeared phasebright with a more rounded appearance (Fig. 2b,c), and the neurites were thinner, with a well-defined growth cone (Fig. 2c), suggesting an altered interaction with the culture surface. Overall, PC12 cells receiving NGF + LPS were more similar to primary neurons in culture than cells receiving NGF alone. The presence of LPS also strongly potentiated the number of PC12 cells responding to NGF, as well as the magnitude of their response (Fig. 3). The number of PC12 cells responding to 1 ng/ml NGF with neurites greater than one cell body diameter more than doubled when LPS (1 mg/ml) was also present, increasing from 20.8 ± 4.0 to 42.2 ± 6.1%. After exposure to 1 ng/ml NGF, the number of PC12 cells showing neurites greater than three cell body diameters in length was routinely low, at 1.0 ± 1.6%, but increased prominently to 15.7 ± 3.8%when 1 mg/ml LPS was also pre-
sent. When LPS was increased to 10 mg/ml, no significant further increase in effect was observed. Addition of a monoclonal antibody to 2.5S NGF prevented morphological differentiation by NGF either alone or in combination with LPS (data not shown). PC12 cells that responded to NGF by the development of neurites generally showed a multipolar appearance. While cells with one to six neurites were observed, about 80% of the responding cells had two to four neurites/cell, and this was not affected by the concentration of NGF over a range of 0.1–10 ng/ml (Fig. 4a). However, PC12 cells exposed to NGF in the presence of LPS tended to be unipolar or bipolar, as seen in Fig. 1c, and this was confirmed by examination of the distribution of the number of neurites per responding cell (i.e. at least one neurite; Fig. 4). For example, in the presence of 0.1 ng/ml NGF, 34.6% of responding
Fig. 2. Dose-dependency of NGF-induced outgrowth of neurites from PC12 cells, showing presence of neurites at least one or three cell body diameters (CBD) in length. Data are the mean ± SEM.
S. Lourenssen, M.G. Blennerhassett / Neuroscience Letters 248 (1998) 77–80
79
signalling mechanism is also activated in PC12 cells following the addition of NGF [7], an enhanced turnover of phospholipids may be involved in the morphological changes in the PC12 cells that occur upon the addition of NGF and LPS. LPS may be generated after damage to the cell membrane, and therefore could be involved with the action of NGF in the regulation of inflammatory and immune reactions through its effects on mast cells [5]. Our study has shown that LPS can also strongly potentiate the action of NGF as a neurotrophin. Since sympathetic neurons remain dependent on NGF in the adult [14], LPS may be a naturally occurring factor affecting neural regeneration in the periphery. Further, this interaction may have important functional consequences on neurons at sites of damage or inflammation, through sensitization of nociceptors. Recent evidence has implicated NGF as the major factor mediating inflammatory hyperalgesia, where LPS may act both indirectly through the activation of mast cells or post-ganglionic sympathetic fibres [1,12] and directly through the sensitization Fig. 3. LPS increases the response of PC12 cells to NGF. LPS added to NGF (1 ng/ml) increased the percent of cells with neurites longer than one or three cell body diameters (CBD) compared with NGF alone. Data are the mean ± SEM; *P , 0.05 relative to control.
PC12 cells had three neurites, while 13.5% extended a single neurite (Fig. 4a). Addition of LPS (1 mg/ml) changed this significantly: only 6.5% of cells had three neurites, and 55.8% of cells had one neurite (Fig. 4b). While 24.1% of cells exposed to 1 ng/ml NGF had five neurites, only 6.3% of cells had five neurites when LPS was also present. No cells with more than four neurites were detected under those conditions. To determine whether LPS would maintain the differentiated state, the media was changed 48 h after addition of NGF and LPS, and media alone or containing LPS without NGF was added. After 24 h, significant retraction of neurites had occurred, which was not affected by the presence of LPS (data not shown). Therefore, the more strongly differentiated state that results from the addition of NGF + LPS requires the presence of NGF for its maintenance. The effects of compounds related to LPS were also tested. Neither lysophosphatidylcholine nor lysophosphatidylinositol affected the morphology of PC12 cells, indicating the absolute requirement for the serine group. Sphingomyelin was also tested, since NGF-induced sphingomyelin metabolism leads to the production of ceramide, implicated in growth arrest and differentiation [8], but no effect was seen. Therefore, the effects of LPS seem specific, and are not a general outcome of the presence of a lipid molecule. Our results provide evidence that LPS can modulate the effects of NGF in a neuronal cell type. While it is possible that LPS acts at the cell surface to facilitate the interaction between p75 and trkA, addition of LPS and NGF to mast cells led to an increased activation of phospholipase C and phosphoinositide hydrolysis [3]. Since the phosphoinositide
Fig. 4. Distribution of neurite number among PC12 cells responding to either NGF alone (A) or NGF + LPS (B). Scale bars, mean ± SEM; *P , 0.05 relative to NGF alone.
80
S. Lourenssen, M.G. Blennerhassett / Neuroscience Letters 248 (1998) 77–80
of sensory neurons. Further studies in vivo may elucidate a potential role of LPS in neuronal sensitization after injury. [1] Andreev, N.Y., Dimitrieva, N., Koltzenburg, M. and McMahon, S.B., Peripheral administration of NGF in the adult rat produces a thermal hyperalgesia that requires the presence of sympathetic post-ganglionic neurons, Pain, 63 (1995) 109–115. [2] Barker, P.A. and Shooter, E.M., Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to trkA on PC12 cells, Neuron, 13 (1994) 203–215. [3] Bellini, F., Toffano, G. and Bruni, A., Activation of phosphoinositide hydrolysis by nerve growth factor and lysophosphatidylserine in rat peritoneal mast cells, Biochim. Biophys. Acta, 970 (1988) 187–193. [4] Bruni, A., Bigon, E., Boarato, E., Mietto, L., Leon, A. and Toffano, G., Interaction between nerve growth factor and lysophosphatidylserine on rat peritoneal mast cells, FEBS Lett., 138 (1982) 190–192. [5] Bruni, A., Monastra, G., Bellini, F. and Toffano, G., Autocoid properties of lysophosphatidylserine, Prog. Clin. Biol. Res., 282 (1988) 165–179. [6] Chao, M.V., Neurotrophin receptors: a window into neuronal differentiation, Neuron, 9 (1992) 583–593. [7] Contreras, M.L. and Guroff, G., Calcium-dependent nerve
[8]
[9]
[10]
[11] [12]
[13]
[14]
growth factor-stimulated hydrolysis of phosphoinositides inPC12 cells, J. Neurochem., 48 (1987) 1466–1472. Dobrowsky, R.T., Jenkins, G.M. and Hannun, Y.A., Neurotrophins induce sphingomyelin hydrolysis, J. Biol. Chem., 270 (1995) 22135–22142. Greene, L.A., Aletta, J.M., Rukenstein, A. and Green, S.H., PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation, Methods Enzymol., 147 (1986) 207–216. Horigome, K., Pryor, J.C., Bullock, E.D. and Johnson, E.M., Mediator release from mast dells by nerve growth factor. Neurotrophin specificity and receptor mediation, J. Biol. Chem., 268 (1993) 14881–14887. Levi-Montalcini, R., The nerve growth factor: thirty five years later, EMBO J., 6 (1987) 1145–1154. Lewin, G.R., Rueff, A. and Mendell, L.M., Peripheral and central mechanisms of NGF-induced hyperalgesia, Eur. J. Neurosci., 6 (1994) 1903–1912. Mazurek, N., Weskamp, G., Erne, P. and Otten, U., Nerve growth factor induces mast cell degranulation without changing intracellular calcium levels, FEBS Lett., 198 (1986) 315–320. Ruit, K.G., Osborne, P.A., Schmidt, R.E., Johnson, E.M. Jr. and Snider, W.D., Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse, J. Neurosci., 10 (1990) 2412–2419.