Nerve Growth Factor (NGF)-induced Calcium Influx and Intracellular Calcium Mobilization in 3T3 Cells Expressing NGF Receptors*

The neurotrophins have been implicated in the acute regulation of synaptic plasticity. Neurotrophin-stimulated presynaptic calcium uptake appears to play a key role in this process. To understand the mechanism of neurotrophin-stimulated calcium uptake, the regulation of calcium uptake and intracellular mobilization by nerve growth factor (NGF) was investigated using NIH 3T3 cells stably transfected with either the high affinity NGF receptor p140 trk (3T3-Trk) or the low affinity NGF receptor p75NGFR(3T3-p75). In 3T3-Trk cells, NGF increased both calcium uptake and intracellular calcium mobilization. In 3T3-p75 cells, NGF increased calcium uptake but not intracellular calcium mobilization. K-252a alone increased intracellular calcium in 3T3-Trk cells but not in 3T3-p75 cells. Nifedipine, an inhibitor of calcium uptake throughl-type calcium channels, inhibited the action of NGF on both 3T3-Trk cells and 3T3-p75 cells, indicating that both p140 trk and p75NGFR receptors are linked to nifedipine-sensitive l-type calcium channels. These studies show that either NGF receptor will support increases in intracellular calcium but that p140 trk does so by increasing both uptake and mobilization, whereas p75NGFR does so by increasing uptake only.

NGF, the first discovered and best characterized member of this family, is required for the survival and development of sympathetic and sensory neurons in the peripheral nervous system (1,2). It also acts on specific populations of neurons in the central nervous system (3)(4)(5) and on cells of the adrenal medulla (6,7).
Two classes of neurotrophin receptors have been identified, usually designated high affinity receptors and low affinity receptors. Many of the biological activities of the neurotrophins are mediated by their binding to and activation of high affinity receptor tyrosine kinases (8). These receptors are encoded by the trk gene family. NGF preferentially binds to p140 trk , BDNF and NT-4/5 to p145 trkB , and NT-3 to p145 trkC (9).
The low affinity neurotrophin receptor p75 NGFR binds all the members of the neurotrophin family with various affinities (3). There is increasing evidence for a role of p75 NGFR in neurotrophin actions, possibly to increase local concentrations of neurotrophin, to alter the specificity of binding to the trk receptors, and/or to enhance the activity of trks (16,17). Most recently, p75 NGFR has been implicated in the activation of the sphingomyelin cycle (18), the migration of Schwann cells (19), the retrograde transport of BDNF and NT-4/5 (20), and the promotion of neuronal apoptosis (21,22).
A great deal of evidence suggests that calcium levels are involved in the long term mechanism(s) by which NGF acts on its target cells. Clearly, neuronal survival requires adequate intracellular calcium levels (23) and, given appropriate calcium levels, the survival of certain classes of neurons becomes neurotrophin-independent (24). Equally clearly, the protection of neurons against environmental insults, such as hypoglycemia or ischemia, depends upon preventing the marked increases in intracellular calcium that attend these insults. In many systems it has been shown that neurotrophins can prevent these increases (25).
More recently it has been shown that the acute actions of the neurotrophins also involve adjustment of calcium levels. It has been found that the addition of BDNF to cultures of Xenopus neuromuscular junctions increases both the spontaneous and the evoked potentials at those synapses (26). Such increases are caused by increased release of neurotransmitter and are the basis of changes in synaptic strength leading to long term potentiation, which, many think, underlies the ability of the brain to store information. And these neurotrophin-dependent changes in neurotransmitter release are dependent, in turn, on increases in presynaptic calcium uptake (27). Thus, it seems likely that the uptake of calcium is a crucial element in both the long and the short term actions of the neurotrophins.
A possible model for these neurotrophin-dependent increases in calcium uptake may be the PC12 cell (28,29). Previous studies have shown that NGF causes increases in calcium uptake into PC12 cells (30) and that this increased uptake seemed to be mediated by unique calcium channels (31). It also appeared that an NGF-induced phosphorylation, perhaps of the calcium channel itself, is required for this increased uptake (31). The strength of the NGF-induced increase in calcium uptake is dependent on the intracellular calcium concentration (32); calcium uptake is greater in cells depleted of calcium and weaker in cells in which calcium levels are raised. Protein kinase C appears to participate in the process of NGF-induced calcium uptake (33). K-252a, a kinase inhibitor that blocks the actions of NGF on PC12 cells (34), mimics the action of NGF on calcium uptake (30,35) and may employ the same signaling elements as does NGF, including protein kinase C and, perhaps, the high affinity NGF receptor itself.
In a previous study it was shown that NGF induced an increased uptake of calcium into 3T3 cells stably transfected with either p140 trk or p75 NGFR (36). In the present study we used confocal microscopy to investigate further the alterations in calcium levels produced by NGF in these two separate cell populations.

EXPERIMENTAL PROCEDURES
Materials-NGF and rat collagen type II were purchased from Collaborative Biomedical Products (Bedford, MA). BDNF was obtained from Genzyme (Cambridge, MA). K-252a was kindly provided by Dr. Y. Matsuda (Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan). The monoclonal anti-phosphotyrosine antibody 4G10 was a product of Upstate Biotechnology, Inc. (Lake Placid, NY). The polyclonal anti-TrkA (C14) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and polyclonal anti-p75 antibody was obtained from Promega (Madison, WI).
Cell Culture and Transfection-3T3 transfectants were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 100 g of streptomycin and 100 units of penicillin (Life Technologies, Inc.) per ml. The pCMV-trkA expression plasmid (36) and the pCMV5-p75 expression plasmid (kindly provided by Dr. Moses Chao) were transfected separately into NIH 3T3 cells by the calcium phosphate method as described previously (36). The stable clones WT11, L2, and L9 were selected in 0.5 mg/ml G418 and were used in the present studies. For WT108, human trkA cDNA was subcloned into the EcoRI site of the retroviral vector pLXSN (37) and transfected into NIH 3T3 cells according to methods previously described (38). For kinase-deficient trkA clones, KD215 and KD217, a mutation of Lys-538 to Asn (K538N) was introduced into pLXSN-trkA with the Quickchange site-directed mutagenesis kit (Stratagene).
Immunoblotting and Immunoprecipitation-Cells were treated with a lysis buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% deoxycholate, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium vanadate, and 50 mM sodium fluoride at 4°C for 30 min. Insoluble material was removed by centrifugation, and equal amounts of lysates were used for either immunoblotting or immunoprecipitation according to the method previously described (39). Specific proteins were detected with a chemiluminescence protein detection kit (Pierce).
Measurement of Intracellular Calcium-3T3 transfectant cells were plated into collagen-and polylysine-coated two-well chambers (Nalgene) 1 day before each experiment. The cells were loaded with 4 M Fluo-3-AM (Molecular Probes, Eugene, OR) for 1 h at 37°C with slight modifications of the methods previously described (32). The cells were then washed twice with 1-ml portions of fresh medium and immediately used for experiments. The fluorescence of intracellular Fluo-3 was quantitated by confocal laser scanning fluorescence microscopy (Leica TCS4D, Leica Lasertechnik Heidelberg, Germany) using excitation and emission wavelengths of 488 and 525, respectively. Gray scale images with 0 -255 steps were collected at different time points before and up to 10 min after the addition of NGF by using a 512 ϫ 512 pixel format and archived as tiff image files for later analysis. The intensity of the fluorescence in individual cells was measured using Leica quantitation software. For each treatment, the relative intensity of three or more typical cells was measured, and the mean value of the fluorescence per unit area of the cell was calculated.
RT-PCR Analysis of Calcium Channel Subunit Transcripts-Total RNA was isolated from NIH 3T3 cells and PC12 cells by RNA-STAT-60 (Tel Test, Inc., Friendswood, TX). RT-PCR reactions were carried out by Superscript One-step RT-PCR system (Life Technologies, Inc.) for 35

FIG. 1. Characterization of 3T3-Trk and 3T3-p75 transfectants.
A, Western blot analysis of 25-g portions of lysates from parent 3T3 cells, 3T3-Trk clones, WT11 and WT108, and 3T3-p75 clones, L2 and L9, using anti-TrkA and anti-p75 antibodies, respectively. B, immunoprecipitation (IP) and Western blot analysis of 1-mg portions of lysates from control and from NGF-treated 3T3-Trk clones, WT11 and WT108, and from 3T3-kinase-deficient Trk clones, KD215 and KD217, using anti-Trk antibody C-14 for immunoprecipitation and anti-phosphotyrosine antibody 4G10 for blotting. The basal levels of Trk receptor were determined by stripping the membrane and reprobing with anti-Trk antibody C-14. C, immunoprecipitation and Western blot analysis of 1-mg portions of lysates from 3T3-Trk clones, WT11 and WT108, treated with NGF and K-252a. The cells were preincubated, where indicated, with either 200 or 500 nM K-252a for 30 min before treatment with 100 ng/ml NGF for 5 min. The lysates were immunoprecipitated with anti-Trk antibody C-14 and immunoblotted with anti-phosphotyrosine antibody 4G10. The basal levels of Trk receptor were determined by stripping the membrane and reprobing with anti-Trk antibody C-14.

RESULTS
The expression of human p140 trk and of p75 NGFR in 3T3-Trk and 3T3-p75 transfectants, respectively, was analyzed by immunoblotting and immunoprecipitation. Both p140 trk and p75 NGFR were highly expressed in the appropriately transfected 3T3 cells but not in the parent cells (Fig. 1A). Indeed, p140 trk is seen as a doublet, as it is in other p140 trk -containing cell lines, the lower band representing an underglycosylated precursor (41)(42)(43). As noted previously (36), the WT 11 and WT 108 clones of 3T3-Trk cells have 3-4-fold more p140 trk receptors than do PC12 cells; the 3T3-p75 clones have about 50% as many p75 NGFR receptors as do PC12.
To examine NGF-induced p140 trk phosphorylation and downstream signaling pathways, WT11 and WT108 cells were treated with 100 ng/ml NGF for 5 min. NGF induced a strong phosphorylation of p140 trk (Fig. 1B) and an activation of mitogen-activated protein kinase (data not shown) in both clones. NGF failed, however, to induce either p140 trk phosphorylation (Fig. 1B) or mitogen-activated protein kinase activation (data not shown) in either KD215 or KD217, clones transfected with a kinase-deficient mutant of p140 trk . K-252a, a tyrosine kinase inhibitor that acts on the Trk family of neurotrophin receptors in PC12 cells (34), blocked NGF-induced p140 trk phosphorylation in both WT11 and WT108 cells (Fig. 1C). These results suggest that transfection of human p140 trk into NIH 3T3 cells results in activation of functional NGF receptors coupled to appropriate downstream signaling pathways.
Previous studies have shown that NGF induced 45 Ca 2ϩ influx into PC12 cells (30) and into both 3T3-Trk and 3T3-p75 cells as well (36). To explore this observation further and to determine the subcellular distribution of the increased intracellular calcium, the calcium-sensitive dye Fluo-3 (44) was used in these studies. In calcium-containing medium, NGF induced an increase in intracellular calcium in both WT11 ( Fig. 2A) and WT108 cells (data not shown). The time of peak increase is about 4 min after NGF treatment (Fig. 2C). The intracellular calcium level began to decrease after about 6 min but remained above basal for at least 10 min after NGF treatment. Nuclear calcium levels were somewhat higher than those in the cytosol.
Recently it has been shown that NGF induces intracellular calcium mobilization in C6 glioma cells transfected with p140 trk receptors (45). In order to examine the effects of NGF on both calcium influx and calcium mobilization, regular calcium-containing medium was replaced with either calcium-free medium or regular medium containing 2 mM EGTA. In WT11 cells in calcium-free medium, NGF also induced an initial increase in intracellular calcium levels (Fig. 2B). The peak was reached in about 3-4 min, and the levels returned to basal in 8 min (Fig. 2D). Ligand-induced increases in intracellular calcium levels in calcium-free medium have been attributed to mobilization of intracellular stores. The persistent increased levels above basal seen in calcium-containing, but not in calcium-free, medium are thought to be due to uptake from extracellular sources. This observation suggests that activation of p140 trk by NGF can induce both calcium influx and release of calcium from internal stores.
In order to define the role of the p140 trk receptor in this process, two kinase-deficient clones, KD215 and KD217, were used. NGF did not induce any increase in intracellular calcium concentrations in KD217 cells (Fig. 3A). Preincubation with 500 nM K-252a for 30 min blocked the NGF-induced increase of intracellular calcium in both WT108 cells (Fig. 3, B and C) and WT11 (data not shown), showing that p140 trk phosphorylation is required to support increased intracellular calcium levels.
In the 3T3-p75 clone L9 (Fig. 4A), NGF also induced a significant increase in intracellular calcium levels. The peak of the increase was at about 6 min after NGF treatment (Fig. 4C). There appeared to be an increase in nuclear calcium levels in these cells as well, but the increase was no greater than that seen in the cytoplasm.
In 3T3-p75 cells, NGF induced an increase in intracellular calcium in the presence of extracellular calcium but had no effect in the absence of extracellular calcium (Fig. 4, B and D). This suggests that activation of p75 NGFR supports only calcium uptake but not calcium mobilization from intracellular stores. Preincubation with K-252a did not block the NGF-induced increase in intracellular calcium in 3T3-p75 cells (data not shown).
Signaling by the p75 NGFR appears to involve the sphingomyelin cycle and the second messenger ceramide (18). The addition of C 2 -ceramide to 3T3 cells resulted in an increase in intracellular calcium (Fig. 5A). Consistent with this, ceramide treatment produced no increase in intracellular calcium in calcium-free medium (Fig. 5B). So the addition of ceramide FIG. 3. Lack of NGF-induced changes in intracellular calcium levels in the kinase-deficient 3T3-Trk clone, KD217, and in the 3T3-Trk clone, WT108, treated with K-252a. Cells were loaded as described in Fig. 2, incubated in calcium-containing medium, and treated with 100 ng/ml NGF. A, KD217 cells; B, WT108 cells; C. WT108 cells preincubated with 500 nM K-252a for 30 min before the addition of NGF. appears to mimic the action of NGF on p75 NGFR ; it produces an increase in calcium uptake but no increase in calcium mobilization.
Previous experiments (30,35) have shown that K-252a alone mimics the actions of NGF on 45 Ca 2ϩ uptake, as it does on neurotransmitter release (46), in PC12 cells, and that uptake appears to be mediated by the p140 trk receptor (35). Treatment of 3T3-Trk cells with K-252a produced a marked increase in calcium levels in the cells (Fig. 6A) and that increase appeared, as does the increase produced by NGF in these cells, to be comprised of an increase in calcium uptake and an increase in calcium mobilization as well, since the increase in calcium-free medium was less than that in complete medium and returned to base line readily (Fig. 6B). Treatment of 3T3-p75 cells with K-252a in calcium-containing medium produced no increase in calcium levels (Fig. 6C). Thus, the actions of K-252a on these cells appears to be mediated by the p140 trk receptor, as it does in PC12 cells (35).
Since recent data indicate that NGF stimulates calcium uptake through L-type calcium channels in PC12 cells, 2 L-type calcium channels were sought in these 3T3 transfectants. PCR analysis showed the presence of transcripts for calcium channel subunits ␣1A, ␣1B, and ␣1C in both 3T3 cells and PC12 (Fig. 7). This last, ␣1C, is the ␣ subunit found in L-type calcium channels. As further proof, it was shown that BayK 8644, an L-type calcium channel agonist, stimulates calcium uptake into these cells and that this uptake is inhibited by nifedipine, an L-type channel antagonist (Fig. 8, A and B). When nifedipine was added to cells stimulated with NGF, inhibition was seen with both 3T3-Trk (Fig. 9, A and B) and 3T3-p75 cells (Fig. 10,  A and B), L-type indicating that binding to either p140 trk receptors or p75 NGFR receptors will activate nifedipine-sensitive L-type calcium channels. DISCUSSION The effect of NGF on calcium uptake, especially in PC12 cells, has been a contentious subject for some 2 decades. It was first reported in 1978 (47) that NGF treatment produces a small increase of calcium release from PC12 cells. However, this observation was not supported by subsequent studies (48). More recently, the direct effects of NGF on the intracellular calcium levels of NGF-responsive cells have been reinvestigated and have led to the finding that NGF produces a small FIG. 4. NGF-induced changes in intracellular calcium levels in 3T3-p75 cells. L9 cells were loaded as described in Fig. 2, incubated in either calcium-containing or calcium-free medium, and treated with 100 ng/ml NGF. A, calciumcontaining medium; B, calcium-free medium; C, measurement of the fluorescence of cells in A; D, measurement of the fluorescence of cells in B. and rapid increase in intracellular calcium concentrations in PC12 cells (49,50) and that certain of the effects of NGF depend upon the presence of extracellular calcium (51,52).
One of the effects of NGF on PC12 cells is to induce catecholamine release, and this release is dependent upon the presence of extracellular calcium (46). By using an assay that measures the uptake of radioactive calcium into PC12 cells, it was further shown that NGF induced a small but rapid increase in the uptake of calcium (30). The increase of calcium uptake is maximal after 5 min of NGF treatment and gradually disappears after 15 min. This observation was further supported by studies using the fluorescent dye, Fluo-3 (32). Those experiments showed that NGF treatment of PC12 cells produces a rapid and transient uptake of divalent cations and an increase in their intracellular level as well. Most significant, this increase was stronger when the calcium levels of the cells were low and weaker when it was high. This suggests that the effects of NGF are to modulate calcium levels in either direction, depending on the needs of the cells at the time.
In order to identify potential calcium channels involved in NGF-induced calcium uptake into PC12 cells, a number of calcium channel blockers were used (31). Nifedipine, a blocker of L-type calcium channels, partially inhibited NGF-induced calcium uptake, suggesting that L-type calcium channels may be at least partly responsible for the NGF-induced calcium uptake. This suggestion has been confirmed through whole cell patch clamping studies. 2 Both p140 trk and p75 NGFR are present on PC12 cells. Experiments with 45 Ca 2ϩ using 3T3-Trk and 3T3-p75 cells have shown that both of these receptors will support increased calcium uptake (36). These experiments highlight an additional Trk-independent role for the low affinity NGF receptor, p75 NGFR , and may be related to some recent observations on NGF-induced neurotransmitter release; the NGF-induced re-FIG. 5. C 2 -ceramide-induced changes in intracellular calcium levels in parent 3T3 cells. 3T3 cells were loaded as described in Fig. 2, incubated in either calcium-containing or calcium-free medium, and treated with 10 M C 2 -ceramide. A, calcium-containing medium; B, calcium-free medium. lease of dopamine from striatal neurons is mediated by p75 NGFR (53), and NGF enhances the depolarization-evoked release of glutamate and acetylcholine from visual cortex synaptosomes through p75 NGFR as well as through p140 trk (54).
The present studies also show that both receptors will mediate increases in intracellular calcium levels. NGF induces both increased calcium uptake and increased calcium mobilization through p140 trk but only increased calcium uptake through p75 NGFR . In this regard it should be noted that, although neurotrophin-mediated neurotrophin release through p140 trk is supported by the mobilization of intracellular calcium stores (55), more recent data show that p75 NGFR also can promote neurotrophin-mediated neurotrophin release (56). Since the present data show that p75 NGFR does not support the mobilization of intracellular calcium stores, it is reasonable to conclude that NGF-induced calcium uptake from the extracellular compartment can also support neurotrophin-induced neurotrophin release.
The response of p140 trk requires intracellular signaling, since kinase-deficient mutants do not support either function. The response of p75 NGFR also appears to require intracellular signaling, since C 2 -ceramide produces an increase in calcium uptake, and it is known that the p75 NGFR can signal through the ceramide pathway (18). The ability of p140 trk to promote the mobilization of intracellular calcium is consistent with what is known about its signaling, i.e. its ability to activate phospholipase C␥ (12), which produces inositol 1,4,5-trisphosphate, which, in turn, activates one of the receptors controlling intracellular calcium release. No such mobilizing function has been ascribed to p75 NGFR .
The data suggest that both p140 trk and p75 NGFR support increases in nuclear calcium as well as in cytoplasmic calcium. This is significant because it has recently been shown that increases in nuclear calcium levels have different biological consequences than do increases in cytoplasmic calcium (57).
It is important to know what mechanism is activated by NGF to bring calcium into the cell, because it is clear that the mechanism by which calcium enters determines its actions within the cell (58). In the case of the receptors studied here both appear to be linked to L-type calcium channels. L-type channels are clearly present on the cells, and an antagonist of L-type channels inhibits the action of NGF through either of its receptors. Although this may only be true in this cell type, it is important to note that nifedipine also inhibits, at least partially, the uptake of 45 Ca 2ϩ into PC12 cells (31), and the application of NGF or of BDNF to PC12 cells produces increases in voltage-activated calcium currents through L-type channels when analyzed by whole cell patch clamping. 2 In summary, neurotrophins are critical for neuronal survival, neuronal differentiation, neuronal protection, and synaptic remodeling. For many of these long and short term functions of the neurotrophins, the control of calcium levels is crucial. In particular, the neurotrophin-mediated presynaptic uptake of calcium, which leads to the neurotrophin-stimulated presynaptic release of neurotransmitter, appears to be a key element in synaptic plasticity and long term potentiation. The detailed mechanism(s) by which the neurotrophins stimulate calcium uptake and modulate intracellular calcium levels provides an intriguing avenue for study.  10. Effect of nifedipine on NGF-induced changes in intracellular calcium levels in 3T3-p75 cells. L9 cells were loaded as described in Fig. 2, incubated in calcium-containing medium, and preincubated with or without 10 M nifedipine for 30 min before treatment with 100 ng/ml NGF. A, fluorescence of L9 cells without nifedipine; B, fluorescence of L2 cells with nifedipine.