01-125 Sigma-AldrichNGF 2.5S Protein, mouse

The uptake of norsalsolinol, a neurotoxin candidate causing parkinsonism-like symptoms, was studied in PC12 cells. The compound was actively taken up by the PC12 cells, with a Km value of 176.24 +/-9.1 microM and a maximum velocity of 55.6 +/- 7.0 pmol/min per mg protein; norsalsolinol uptake was dependent on the presence of extracellular Na+. The uptake of norsalsolinol was sensitive to two dopamine transporter inhibitors, GBR-12909 and reserpine, but was less sensitive to desipramine, a noradrenaline transporter inhibitor. Dopamine competitively inhibited norsalsolinol uptake into PC12 cells with a Ki value of 271.2 +/- 61.6 microM. These results suggest that norsalsolinol is taken up into PC12 cells mainly by the dopamine transporter.

Nerve growth factor (NGF) can influence mast cell development and function in murine rodents by interacting with its receptors on mast cells. We now report the identification of mRNA transcripts of full-length tyrosine kinase-containing trkA, trkB, and trkC neurotrophin receptor genes in HMC-1 human mast cell leukemia cells. Although HMC-1 cells lacked p75 mRNA, they expressed transcripts for the exon-lacking splice variant of trkA (trkAI), truncated trkB (trkB.T1), and truncated trkC. By flow cytometry, HMC-1 cells exhibited expression of TrkA, TrkB, and TrkC receptor proteins containing full-length tyrosine kinase domains. NGF stimulation of HMC-1 cells induced tyrosine phosphorylation of TrkA protein, increased expression of the early response genes c-fos and NGF1-A, and activation of ERK-mitogen-activated protein (MAP) kinase, results which indicate that TrkA receptors in HMC-1 cells are fully functional. Highly purified populations of human lung mast cells expressed mRNAs for trkA, trkB and trkC, whereas preparations of human umbilical cord blood-derived mast cells expressed mRNAs for trkA and trkC, but not trkB. Moreover, preparations of human umbilical cord blood-derived immature mast cells not only expressed mRNA transcript and protein for TrkA, but exhibited significantly higher numbers of chymase-positive cells after the addition of NGF to their culture medium for 3 weeks. In addition, HMC-1 cells expressed mRNAs for NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), the cognate ligands for TrkA, TrkB, and TrkC, whereas NGF and BDNF transcripts were detectable in human umbilical cord blood mast cell preparations. Taken together, our findings show that human mast cells express a functional TrkA receptor tyrosine kinase and indicate that NGF may be able to promote certain aspects of mast cell development and/or maturation in humans. Our studies also raise the possibility that human mast cells may represent a potential source for neurotrophins.

Regulation of heart rate by the sympathetic nervous system involves the release of norepinephrine (NE) from nerve terminals onto heart tissue, resulting in an elevation in beat rate. Nerve growth factor (NGF) is a neurotrophin produced by the heart that supports the survival and differentiation of sympathetic neurons. Here we report that NGF also functions as a modulator of sympathetic synaptic transmission. We determined the effect of NGF on the strength of synaptic transmission in co-cultures of neonatal rat cardiac myocytes and sympathetic neurons from the superior cervical ganglion (SCG). Synaptic transmission was assayed functionally, as an increase in the beat rate of a cardiac myocyte during stimulation of a connected neuron. Application of NGF produced a pronounced, reversible enhancement of synaptic strength. We found that TrkA, the receptor tyrosine kinase that mediates many NGF responses, is expressed primarily by neurons in these cultures, suggesting a presynaptic mechanism for the effects of NGF. A presynaptic model is further supported by the finding that NGF did not alter the response of myocytes to application of NE. In addition to the acute modulatory effects of NGF, we found that the concentration of NGF in the growth medium affects the level of synaptic transmission in cultures of sympathetic neurons and cardiac myocytes. These results indicate that in addition to its role as a survival factor, NGF plays both acute and long-term roles in the regulation of developing sympathetic synapses in the cardiac system.

The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10(-9) M and as early as 4 h. The phosphatidylinositol 3' (PI3')-kinase inhibitors wortmannin (at concentrations of 10(-8) M) and LY294002 (10(-6) M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70S6K) inhibitor rapamycin (10(-8) M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3'-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10(-5) M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.

Tyrosine autophosphorylation controls the catalytic and signaling activities of the neurotrophin receptors, the Trks. To analyze the regulation of distinct tyrosine sites, we generated a panel of antibodies that report the phosphorylation state of individual tyrosines within the Trk cytoplasmic domain. Using pheochromocytoma-derived cell lines, we show that individual tyrosines within the nerve growth factor receptor TrkA are phosphorylated in a non-coordinate fashion following receptor activation. The non-coordinate response of these tyrosines reflects their separate functions in regulating the catalytic and signaling activities of Trk receptors. The differential utilization of distinct sites on Trk receptor tyrosine kinases suggests that the receptor can specify both the timing and the nature of neurotrophin-stimulated signal transduction pathways. Moreover, we show that these Trk autophosphorylation sites, which have hitherto been mapped and characterized only in non-neuronal cell lines, are activated in normal neurons in response to ligand stimulation.

Plasmalopsychosine, a characteristic fatty aldehyde conjugate of beta-galactosylsphingosine (psychosine) found in brain white matter, enhances p140trk (Trk A) phosphorylation and mitogen-activated protein kinase (MAPK) activity and as a consequence induces neurite outgrowth in PC12 cells. The effect of plasmalopsychosine on neurite outgrowth and its prolonged activation of MAPK was similar to that of nerve growth factor (NGF), and the effect was specific to neuronal cells. Plasmalopsychosine was not capable of competing with cold chase-stable, high affinity binding of NGF to Trk A, indicating that plasmalopsychosine and NGF differ in terms of Trk A-activating mechanism. Tyrosine kinase inhibitors K-252a and staurosporine, known to inhibit the neurotrophic effect of NGF, also inhibited these effects of plasmalopsychosine, suggesting that plasmalopsychosine and NGF share a common signaling cascade. Plasmalopsychosine prevents apoptosis of PC12 cells caused by serum deprivation, indicating that it has "neurotrophic factor-like" activity. Taken together, these findings indicate that plasmalopsychosine may play an important role in development and maintenance of the vertebrate nervous system.

Inherited activating mutations of Ret, a receptor tyrosine kinase, predispose to multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma. To investigate the effects induced by acute stimulation of Ret, we transfected both PC12 and NIH 3T3 cells with a molecular construct in which the ligand-binding domain of the epidermal growth factor receptor was fused to the catalytic domain of Ret. Acute stimulation of the chimeric receptor induced PC12 cells to express a neuronal-like phenotype. Moreover, we introduced the dominant mutation, responsible for the multiple endocrine neoplasia type 2B, in the catalytic domain of the Ret chimera. Expression of the mutant chimera, in the absence of ligand stimulation, induces the PC12 cells to acquire a flat morphology with short neuritic processes and transforms the NIH 3T3 cells. Stimulation of the mutant chimera with epidermal growth factor causes a drastic overgrowth of long neuritic processes, with the induction of the suc1-associated protein tyrosine phosphorylation in PC12 cells and higher transforming efficiency in NIH 3T3 cells. These data indicate that the gain-of-function MEN2B mutation does not abrogate ligand responsiveness of Ret and suggest that the presence of Ret ligand could play a role in the pathogenesis of the MEN2B syndrome.

The cellular redox state is thought to play an important role in a wide variety cellular signaling pathways. Here, we investigated the involvement of redox regulation in the nerve growth factor (NGF) signaling pathway and neuronal differentiation in PC12 cells. N-acetyl-L-cysteine (NAC), which acts as a reductant in cells both by its direct reducing activity and by increasing the synthesis of the cellular antioxidant glutathione, inhibited neuronal differentiation induced by NGF or by the expression of oncogenic ras in PC12 cells. NAC suppressed NGF-induced c-fos gene expression and AP-1 activation. These results suggest that neuronal differentiation and NGF signaling are subject to regulation by the cellular redox state. NAC also suppressed the NGF-induced activation of mitogen-activated protein kinases (MAPKs) and decreased the amount of tyrosine phosphorylation of MAPKs. The suppression of MAPK by NAC was independent of glutathione synthesis. In parallel with the suppression of MAPK, the activation of MAPK kinase kinase activity was also suppressed in the presence of NAC. In contrast, NGF-induced activation of Ras was not inhibited by NAC. The inhibitory effect of NAC on the MAPK cascade was independent of transcription and translation. Thus, NAC suppresses NGF-induced neuronal differentiation by uncoupling the signal transduction from Ras to the MAP kinase cascade in PC12 cells.