Characteristics of the K-252a-induced increase in calcium uptake in PC12 cells

K-252a treatment produced a 30–50% increase in the uptake of radioactive calcium by PC12 cells within 3–4 minutes. The increase in uptake was partially blocked by inhibitors of voltage-operated calcium channels, such as nifedipine, but not by inhibitors of receptor-operated calcium channels, such as nickel or suramin. Introduction of phosphatase 2A into the cells completely blocked the effect of K-252a. Longterm treatment of the cells with either K-252a or with nerve growth factor blocked the subsequent actions of either K-252a or nerve growth factor on calcium uptake, but neither altered the subsequent action of the L-channel agonist Bay K 8644 on calcium uptake. Calcium uptake was not stimulated by K-252a in PC12nnr, cells that have little or no high-affinity nerve growth factor receptors; cells expressing increased levels of high-affinity nerve growth factor receptors showed a response to K-252a comparable to that seen in parent PC12. The data suggest that the increased uptake of radioactive calcium produced by K-252a is mediated by a mechanism very similar to that serving the increased calcium uptake produced by nerve growth factor. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells

The PC12 clone is a line of rat pheochromocytoma cells that undergoes neuronal differentiation in the presence of NGF protein. In the absence of NGF, PC12 cells are electrically inexcitable, while after several weeks of NGF treatment they develope Na+ action potentials. Past estimates made by measuring binding of 3H-saxitoxin (STX) indicate that NGF treatment brings about a large increase in Na channel density that is of sufficient magnitude to account for the induction of excitability. We have now used 22Na uptake to measure the Na permeability of PC12 cells before and after long-term NGF treatment. Treatment with NGF does not change the resting Na+ permeability. The alkaloid toxins veratridine and batrachotoxin (BTX) and scorpion toxin were used to activate Na channels. Such studies demonstrate that these toxins induce TTX-sensitive Na uptake in both NGF-treated and untreated cells and reveal differences in functional Na channel numbers per cell and per unit of membrane area that are similar to those found in the STX binding studies. On the other hand, affinities for drugs that activate these channels are not affected by NGF treatment. We also find that NGF-treated PC12 cells contain a population of Na channels with low affinity for TTX. These channels account for 5-20% of total BTX or veratridine-stimulated flux. Thus, NGF has 2 effects regarding the Na channels of PC12 cells: it increases the number of functional Na channels that otherwise behave similarly to those present before NGF treatment, and it induces the presence of TTX-resistant Na channels. These findings indicate that the PC12 model system may serve to study the developmental regulation of Na channel expression and properties.     

Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells

PC12 cells are a pheochromocytoma cell line that can be made to differentiate into sympatheticlike neurons by nerve growth factor (NGF). An essential component of the NGF-induced differentiation is the development of action potentials and sodium channels. Using whole-cell clamp we have confirmed that NGF produces a 5- to 6-fold increase in sodium channel density. The sodium channels induced by NGF are not different from those in cells not treated with NGF and are similar to those in other cell types. Basic fibroblast growth factor (FGF), another growth factor that causes PC12 cells to differentiate into sympathetic-like neurons, also produces a 5- to 6-fold increase in sodium current density with channels indistinguishable from those in PC12 cells treated and not treated with NGF. Basic FGF produces the same or somewhat larger increase in sodium channel density but much less neurite outgrowth. In contrast, epidermal growth factor does not produce neurite outgrowth but induces a small, reproducible increase in sodium channel density. Cyclic AMP produces spike-like processes but not neurites and results in a decrease in sodium current and sodium current density. Dexamethasone, a synthetic glucocorticoid, inhibits the increase in sodium current and sodium current density but does not antagonize the neurite outgrowth induced by NGF. Thus, although the increase in sodium channel expression induced by NGF and basic FGF parallels the changes in morphology that lead to neurite outgrowth, it clearly does not depend on them. The results show that different aspects of neuronal differentiation might be independently regulated by the microenvironment.     

EGF in combination with depolarization or cAMP produces morphological but not physiological differentiation in PC12 cells

In response to nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) receptor activated Ras/extracellular signal-regulated kinase (ERK) signaling, PC12 cells undergo a prototypical neuronal differentiation program, characterized by neurite extension and upregulation of voltage-gated ion channels. The epidermal growth factor (EGF) receptor also activates Ras/ERK signaling, but produces proliferation instead of differentiation. In the presence of depolarizing concentrations of KCI, however, EGF elicits neurite outgrowth through the synergistic actions of the Ras/ERK and cAMP signaling pathways. To assess if EGF and KCI/cAMP elicit the same suite of differentiation events as does NGF and bFGF, we used patch clamp recording to determine if EGF in the presence of KCI or a cAMP agonist also induced physiological differentiation as defined by upregulation of ion channels. Chronic NGF treatment of PC12 cell cultures elicited robust morphological differentiation, a threefold increase in mean calcium channel current density, and an eightfold increase in mean sodium channel current density. Sibling cultures chronically treated with EGF in the presence of high KCI or a cAMP agonist also displayed morphological differentiation, but had calcium channel current densities which were no larger than untreated, undifferentiated cells. Additionally, the increase in mean sodium channel current density induced by EGF in the presence of KCI or cAMP was no greater than the increase observed with EGF alone. Thus, although EGF in the presence of KCI or cAMP is sufficient to induce morphological differentiation as defined by neurite outgrowth, synergism of the Ras/ERK and cAMP/PKA signaling pathways is not sufficient to promote the fully physiologically differentiated PC12 phenotype. J. Neurosci. Res. 47:16–26, 1997. © 1997 Wiley-Liss, Inc.     

Nerve growth factor-induced neuronal differentiation is accompanied by differential induction and localization of the amyloid precursor protein (APP) in PC12 cells and variant PC12S cells

PC12 cells and the morphological variant PC12S cells in culture were examined by immunochemical methods for the presence of the amyloid precursor protein (APP), before and after treatment with the nerve growth factor (NGF). In untreated PC12, untreated PC12S and in NGF-treated PC12 cells, APP was localized in the cytoplasm, whereas in NGF-treated PC12S cells, APP was localized at growth cones, processes and cytoplasm. In PC12 cells, three major forms of APP (695 and 751/770) were detected by Western blot. After NGF treatment, only the level of APP 695 was increased. Immunoprecipitation studies in PC12 cells revealed six protein species, corresponding to immature and mature forms of each of the three APP 695, 751 and 770 proteins. Addition of NGF increased the synthesis of the immature and mature forms of APP695. In PC12S cells, only the higher molecular weight forms of APP (751/770) were detected by both Western blot and immunoprecipitation. Addition of NGF had no effect on their levels. In both cell types, the level of the secreted form of APP showed a significant transient increase after NGF treatment. These results suggest that NGF can differentially regulate the molecular forms of APP and the localization of APP within the cell.     

Role of protein tyrosine phosphorylation in the NGF response

The role of protein tyrosine phosphorylation in the response of PC12 cells to NGF was investigated by using a variety of agents which affect NGF-induced neurite outgrowth. K-252a, a kinase inhibitor, was previously found to selectively inhibit many of the actions of NGF on PC12 cells. In the present study, it was shown to inhibit NGF-induced protein tyrosine phosphorylation. However, sphingosine, an inhibitor of protein kinase C and NGF-induced differentiation of PC 12 cells, did not alter the phosphorylation of proteins on tyrosine stimulated by NGF. Disruption of either actin microfilaments or microtubules also had no effect on NGF-induced protein tyrosine phosphorylation in PC12 cells. The effect of vanadate, an inhibitor of phosphotyrosyl phosphatases, on the differentiation of PC12 cells was also examined. Vanadate did not promote neurite outgrowth but did stimulate protein tyrosine phosphorylation. Taken together, these results suggest that protein tyrosine phosphorylation is one of the first events in the NGF pathway in PC12 cells but alone is not sufficient to induce morphological differentiation. Finally, the distribution of phosphotyrosine-containing proteins in untreated and NGF-treated cells was examined by immunofluorescence microscopy. The distribution of these proteins was altered by treatment of the cells with NGF and appeared to correlate with the distribution of actin filaments, particularly in growth cones.     

Involvement of protein kinase C in nerve growth factor- and K-252a-stimulated calcium uptake into PC12 cells

Both nerve growth factor (NGF) and K-252a stimulate the uptake of calcium into PC12 cells. Stimulation by either is prevented by pretreatment of the cells with the tumor promoter phorbol 12-myristate 13-acetate (PMA), suggesting an involvement of protein kinase C in the stimulation. The effect of PMA is specific in that the calcium uptake stimulated by either the L-type channel agonist BAY K 8644 or by ATP is not altered in PMA-pretreated cells. An involvement of kinase C is also suggested by the inhibition of NGF- or K-252a-stimulated calcium uptake by the kinase C inhibitors staurosporine and calphostin C. Inhibition by the isoform-specific agents GO 6976 and thymeleatoxin implicates one of the classic calcium-sensitive isoforms of kinase C. The close similarity in the profiles of inhibition of NGF-stimulated and K-252a-stimulated calcium uptake by the various effectors suggests that NGF and K-252a act on calcium uptake through some of the same signaling elements. J. Neurosci. Res. 47:271–276, 1997. © 1997 Wiley-Liss, Inc.     

Nerve growth factor and cyclic AMP: opposite effects on neuroblastoma-substrate adhesion

Increased cellular adhesion has been postulated to be an early event required of neuroblasts undergoing neurite extension during differentiation. Nerve growth factor (NGF) induces neurite extension in a variety of cell types of neural crest origin. In the PC12 rat pheochromocytoma cell line, which has been proposed as a model for precursor cells to sympathetic neurons, NGF and dibutyryl cyclic AMP (dBcAMP) promote both neurite extension and an increased rate of cell-substrate adhesion. Since dBcAMP can substitute for NGF in this enhancement of adhesion rate in the PC12 cell line, cAMP has been suggested as a second messenger for NGF. We have shown that in two nearly diploid adrenergic like human neuroblastoma clones, the KA and SY5Y cell lines, which also extend neurites in response to both NGF and dBcAMP, only NGF enhances cellular adhesion, as defined by an increase in the number of cells cells prelabeled with 35S-methionine which attach to culture dishes at a given time. Incubation with monospecific antibodies directed against murine beta-NGF abolishes the NGF effect on adhesion. The NGF effect on human neuroblastoma is specific insofar as NGF does not facilitate adhesion of two glioma lines. Unlike the results obtained for PC12, in both SY5Y and KA lines, 1 mM dBcAMP decreases the rate of adhesion to levels significantly below those of controls. Adhesiveness of neuroblastoma cells treated with both NGF and dBcAMP is intermediate between that of cells treated with either agent alone. While theophylline mimics the dBcAMP effect, sodium butyrate has no such effect. At 22 degrees C, the effect of NGF on neuroblastoma substrate adhesion is observable within 5 minutes and persists for 2 hours; treatment of the KA line with NGF at 37 degrees C for 24 hours results in a more persistent enhancement of cell adhesion. Furthermore, both SY5Y and KA exhibit different morphologies when challenged with NGF, dBcAMP, or sodium butyrate. This study suggests that NGF and cyclic AMP do not share a common mechanism of action, but can in fact interact antagonistically in an adrenergic like neuroblastoma model system. Furthermore, the results suggest that increased cellular adhesiveness may not be an obligatory prerequisite for neurite extension by neuroblasts in development.     

Acetyl-L-carnitine enhances the response of PC12 cells to nerve growth factor.

We have demonstrated that treatment of rat pheochromocytoma (PC12) cells with acetyl-L-carnitine (ALCAR) stimulates the synthesis of nerve growth factor receptors (NGFR). ALCAR has also been reported to prevent some age-related impairments of the central nervous system (CNS). In particular, ALCAR reduces the loss of NGFR in the hippocampus and basal forebrain of aged rodents. On these bases, a study on the effect of NGF on the PC12 cells was carried out to ascertain whether ALCAR induction of NGFR resulted in an enhancement of NGF action. Treatment of PC12 cells for 6 days with ALCAR (10 mM) stimulated [125I]NGF PC12 cell uptake, consistent with increased NGFR levels. Also, neurite outgrowth elicited in PC12 cells by NGF (100 ng/ml) was greatly augmented by ALCAR pretreatment. When PC12 cells were treated with 10 mM ALCAR and then exposed to NGF (1 ng/ml), an NGF concentration that is insufficient to elicit neurite outgrowth under these conditions, there was an ALCAR effect on neurite outgrowth. The concentration of NGF necessary for survival of serum-deprived PC12 cells was 100-fold lower for ALCAR-treated cells as compared to controls. The minimal effective dose of ALCAR here was between 0.1 and 0.5 mM. This is similar to the reported minimal concentration of ALCAR that stimulates the synthesis of NGFR in these cells. The data here presented indicate that one mechanism by which ALCAR rescues aged neurons may be by increasing their responsiveness to neuronotrophic factors in the CNS.     

An inhibitor of serine proteases,serpinb1a,modulates differentiation of PC12 cells but not cultured neurons

It was shown that serpinb1a is expressed during differentiation of PC12 cells induced by nerve growth factor (NGF). Here, we found that overexpression of serpinb1a in PC12 cells weakly but significantly increases PC12 survival during staurosporine-induced apoptosis. Immunoprecipitation of serpinb1a after its overexpression in PC12 showed that this protein interacts with active caspase-3 in both resting cells and cells that were stimulated by staurosporine. NGF-induced PC12 differentiation resulted in the formation of outgrowths and a considerable increase in caspase-3 activity in 24 h after the beginning of NGF treatment. This increase in the activity of caspase-3 lasted for at least 48 h. Overexpression of serpinb1a in PC12 cells suppressed the growth of neurites during NGF-induced differentiation; this effect was observed only at 48 h. Experiments performed with primary cultures of neocortical and hippocampal neurons showed that serpinb1a overexpression results in relatively weak changes in morphology: serpinb1a decreased the number of secondary dendrites in the cortical and average length of secondary dendrites in hippocampal neurons. The results of the experiments suggests that serpinb1a may interact with caspase-3 and influence the differentiation of PC12 cells but not neuronal cells.     

Role of low-affinity p75 receptor in nerve growth factor-inducible growth arrest of PC12 cells

Mutant PC12 cell clones (PC84 cells) were obtained by transfection with nerve growth factor (NGF) cDNA. These cells secreted active NGF, extended short processes, and proliferated faster than the parental PC12 cells. These features are of great interest because the parental PC12 cells cease proliferation and extend long processes when transfected with NGF cDNA. PC84 cells expressed a high level of acetylcholinesterase activity and neurofilament M, which indicates that PC84 cells were differentiated. The inhibition of TrkA by K252a diminished the short processes of PC84 cells but had no effect on their fast proliferation. The expression level of TrkA in PC84 cells was comparable to that in PC12 cells; whereas that of another NGF receptor, p75, was significantly lower. These data suggest that the decrease of p75 contributed to the continuous growth of PC84 cells, which was confirmed by suppressing p75 activity of PC12 cells with the antisense oligonucleotide of p75 or with anti-p75 neutralizing antibody. The treated cells did not cease proliferation in the presence of NGF and extended short processes. Our results suggest that NGF signaling via TrkA affects the differentiation characteristics of PC12 cells but that an additional signaling via p75 is necessary for the growth arrest of the cells.     

Fibroblast growth factor-induced increases in calcium currents in the PC12 pheochromocytoma cell line are tyrosine phosphorylation dependent

The PC12 rat pheochromocytoma cell line is widely used to study neuronal differentiation by growth factors. In response to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), PC12 cells differentiate into sympathetic-like neurons and become electrically excitable. Using whole cell patch-clamp recording, with barium as a charge carrier, we looked at the effects of bFGF on calcium channel expression as reflected by changes in barium current amplitudes normalized to cell membrane area. Similar to the effect reported for NGF, we show that 7 day treatment with bFGF increased the barium current approximately 4-fold. The largest contributor to the increase in barium current with bFGF treatment is a 6-fold increase in the high threshold voltage activated Ω-conotoxin sensitive barium current. Smaller increases in current produced by bFGF treatment of PC12 cells are observed for the dihydropyridine sensitive and dihydropyridine/conotoxin insensitive currents. The bFGF-induced increases in barium currents are dependent on tyrosine phosphorylation, since the effects of bFGF are blocked by genistein, a tyrosine kinase inhibitor. This system will ultimately be useful in understanding the signaling pathways that control calcium channel expression in response to growth factors. © 1994 Wiley-Liss, Inc.     

Serotonin induces the increase in intracellular Ca2+ that enhances neurite outgrowth in PC12 cells via activation of 5-HT3 receptors and voltage-gated calcium channels

As a neurotransmitter and neuromodulator, serotonin (5-HT) influences neuronal outgrowth in the nervous systems of several species. In PC12 cells, 5-HT is known to have neuritogenic effects, although the signal transduction pathway responsible for these effects is not understood. In this study, we hypothesized that a 5-HT-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) could be involved in mediating the effects of 5-HT. Application of 5-HT to PC12 cells enhanced nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner, and the sensitivity of this neuritogenic effect was increased in differentiated PC12 cells. In accordance, an increase in [Ca(2+)](i) was observed following application of 5-HT in differentiated PC12 cells. This increase was amplified by further NGF treatment. 5-HT-induced increases in [Ca(2+)](i) were inhibited by MDL 72222, a selective 5-HT(3) receptor antagonist, and nifedipine, an L-type calcium channel blocker, but not by ketanserin, a 5-HT(2) receptor antagonist, or thapsigargin, a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase. These pharmacological tests indicated that 5-HT-induced increases in [Ca(2+)](i) are mediated by activation of voltage-gated calcium channels via 5-HT(3) receptors and that 5-HT-induced increases in [Ca(2+)](i) are likely to be independent of activation of 5-HT(2) receptors in PC12 cells. Furthermore, the neuritogenic effect of 5-HT was suppressed by MDL 72222, nifedipine, calmodulin (CaM) inhibitor, and calcineurin inhibitors. Taken together, our results indicate that 5-HT-induced increases in [Ca(2+)](i), which are mediated via 5-HT(3) receptors and L-type calcium channels in PC12 cells, and subsequent activation of CaM and calcineurin enhance NGF-induced neurite outgrowth.     

Fucosylation of glycolipids in PC12 cells is dependent on the sequence of nerve growth factor treatment and adenylate cyclase activation

Synthesis of complex glycosphingolipids was analyzed in cultures of PC12 cells treated with nerve growth factor (NGF) and forskolin, either sequentially or simultaneously. For the sequential treatment, forskolin was added following 15 days of NGF treatment and cultures were continued for 3 additional days. For simultaneous treatment, cells maintained in medium with no additives received both agents and were then incubated for 3 additional days. Sequential NGF/forskolin treatment induced hypertrophy in PC12 cells and produced cells which had more neuron-like characteristics than cells treated with NGF alone. Simultaneous treatment initially accelerated the outgrowth of neurites and then reduced neurite elongation. Sequential treatment of PC12 cells resulted in a greater than 50% increase in the incorporation of fucose into neutral glycolipids and gangliosides compared to treatment with NGF alone. Galactose incorporation was unchanged, indicating that there was no net increase in glycolipid synthesis. The lack of an increase in total glycolipid accumulation was confirmed using monoclonal antibodies reacting with specific fucosylated and nonfucosylated PC12 cell glycolipids. However, TLC immunostaining analysis revealed that sequential NGF/forskolin treatment selectively altered the expression of some neutral glycolipid species. In contrast to the effects of the sequential treatment, simultaneous NGF and forskolin treatment was accompanied by a decrease in the incorporation of fucose and galactose into neutral glycolipids and gangliosides. These findings suggest that the timing of exposure to neuronotrophic factors and agents which activate adenylate cyclase might in part account for developmentally regulated patterns of glycoconjugate expression and provide further evidence that fucosylation of glycolipids may be associated with maturation of sympathetic neurons.     

Nerve growth factor-induced stimulation of alpha-aminoisobutyric acid uptake in PC12 cells: relationship to plasma membrane receptor occupancy

Nerve growth factor (NGF) stimulates the uptake rate of the nonmetabolized amino acid alpha-aminoisobutyric acid (AIB) in the clonal PC12 pheochromocytoma cell line by 40-70%. This effect reaches a maximum after a 1-hour incubation with the hormone and then drops over 50%, reaching a minimum after 4 hours of NGF administration. Longer exposure to the hormone leads to a gradual rise in stimulation, and by 24 hours, the cells regain about 80% of the original 1-hour rate. Results of NGF-binding studies indicate that stimulation of AIB uptake follows closely behind the amount of NGF bound to the low-affinity NGF receptors. Dose-response experiments indicate that full stimulation occurs biphasically. Within the NGF concentration range of 0.1 ng/ml to 1 ng/ml, the AIB uptake rate is 30% of the maximum 1-hour response. At 2.5 ng/ml NGF, the stimulation jumps to about 75% maximal response while 100% response is reached between 5 ng/ml and 50 ng/ml NGF. We suggest that the two states of the NGF plasma membrane receptor on PC12 cells may both be involved in mediating NGF stimulation of AIB uptake.     

Glucocorticoids modulate G-protein α-subunit levels in PC12 cells

Regulation by the synthetic glucocorticoid hormone, dexamethasone, of the levels of several G-protein α-subunits was studied during differentiation in PC12 cells. Similar patterns, although with different magnitudes, were observed in the changes in the levels of αil, αs, and αq induced by the treatments studied, whereas αo differed from the other α-subunits. Thus, nerve growth factor (NGF) treatment increased αil, αs, and αq, and forskolin increased αil and αq, with the increase in αil being greater than the increases in the other two α-subunits after both treatments. The increases in αil, αs, and αq induced by NGF were dependent on signaling through ras, since they did not occur in NGF-treated M17 cells, which express a dominant inhibitory Ha-ras. Treatment of PC12 cells with dexamethasone antagonized the increases in αil, αs, and αq induced by NGF or forskolin, almost completely blocking any changes from control levels. The level of αo also was increased in PC12 cells by treatment with NGF or forskolin, but, in contrast to the other G-protein α-subunits, the response to NGF was not antagonized by dexamethasone in PC12 cells, or by the deficient ras activity in M17 cells. However, ras influenced the alternative splicing that regulates the levels of the two αo subtypes, αo1 and αo2, so they were expressed in a ratio of 1:2 in PC12 cells but 2:1 in ras-deficient M17 cells. These results demonstrated marked, and subtype-selective, influences of dexamethasone on the levels of G-protein α-subunits, an effect that may contribute to the effects of conditions that increase the levels of glucocorticoid hormones, such as stress or certain diseases, on signal transduction processes in brain.     

Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-ζ isoform

To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 μM) resulted in depleting the cells of α, β, δ, and ɛ-PKC isoforms, but had no effect on the expression of the atypical PKC isoform ζ. PC12 cells, which expressed only PKC ζ, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of³H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC ζ (Wooten et al., 1994). To test whether the transient activation of PKC ζ is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5′ coding sequence of PKC ζ attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC ζ comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC ζ.     

Repeated,intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells)

Repeated, intermittent treatment with amphetamine (AMPH) leads to long-term neurobiological adaptations in rat brain including an increased number and branching of dendritic spines. This effect depends upon several different cell types in the intact brain. Here we demonstrate that repeated, intermittent AMPH treatment induces neurite outgrowth in cultured PC12 cells without the requirement for integrated synaptic pathways. PC12 cells were treated with 1 micro M AMPH for 5 min a day, for 5 days. After 10 days of withdrawal, there was an increase in the percentage of cells with neurites ( approximately 30%) and the length of neurites as well as an increase in the level of GAP-43 and neurofilament-M. Neurite outgrowth was enhanced as withdrawal time was increased. Neurite outgrowth was much greater following repeated, intermittent treatment with AMPH compared to continuous or single treatment with AMPH. Pretreatment with cocaine, a monoamine transporter blocker, inhibited the AMPH-mediated increase in neurite outgrowth. Neither NGF antibody nor DA receptor antagonists blocked AMPH-induced neurite outgrowth, demonstrating that AMPH-induced neurite outgrowth is not dependent on endogenous NGF release or DA receptors. Thus we have demonstrated that repeated, intermittent treatment with AMPH has a neurotrophic effect in PC12 cells. The effect requires the action of AMPH on the norepinephrine transporter, and shares characteristics in its development with other forms of sensitization but does not require an intact neuroanatomy.     

Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. I. Stimulation of hydrogen peroxide resistance

The nerve growth factor protein (NGF) regulates neuronal cell death during the development of embryonic sensory and sympathetic neurons in the peripheral nervous system (PNS). NGF protects the rat pheochromocytoma line PC12, a useful model of NGF responsive peripheral neurons, from hydrogen peroxide, which interacts with ferrous iron to generate hydroxyl radicals. Exogenous catalase provides protection, whereas superoxide dismutase (SOD) has no effect on neuronal survival when PC12 cells are challenged with hydrogen peroxide. NGF treatment of PC12 cells increases the activity of catalase. NGF protection from hydrogen peroxide is partially abolished by aminotriazole (Az), a low molecular weight catalase inhibitor. Taken together, these data are consistent with the hypothesis that NGF protects from peroxidative events and consequent cell death via an induction of free radical detoxifying mechanisms, such as catalase activity.