Altered [Ca2+] homeostasis in PC12 cells after nerve growth factor deprivation

[Ca2+]i homeostasis in individual PC12 cells after elevated [K+]o was studied by ratiometric microscopy, during nerve growth factor (NGF) deprivation. A significantly lower number of cells responded with an increased [Ca2+]i in the NGF deprived condition. Moreover, the responding cells were more deficient in regulating their [Ca2+]i back to control levels, after the transient peak. This suggests that differentiated neurons do not traverse the apoptotic program homogeneously with regard to their [Ca2+]i regulation and that NGF deprived PC12 cells have more difficulties to reduce their [Ca2+]i after influx of [Ca2+]o.     

Lumbar disk herniation combined with epidural hematoma. A case report

We examined the protective effect of nicotine against neuronal cell death in the model system of serum- and nerve growth factor (NGF)-free cultures of PC12 cells. Serum deprivation induced massive death of undifferentiated PC12 cells, which was inhibited by the addition of NGF, but not by the addition nicotine (100 microM). Even after PC12 cells had been differentiated by the treatment with NGF, serum and NGF deprivation induced rapid and massive death of these cells. The addition of nicotine (1-100 microM) to the deprivation system prevented such cell death almost completely. The protective effect of nicotine was abolished by hexamethonium or mecamylamine, inhibitors of nicotinic acetylcholine (nACh) receptors. Treatment with reserpine to deplete intracellular catecholamines did not influence the effect. The results suggest that the protective effect of nicotine against neuronal cell death is exerted through nACh receptors.     

Role of Ca2+ in differentiation mediated by nerve growth factor and dibutyryl cyclic AMP in PC12 cells

Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca2+ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca2+ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca2+ influx, and Ca2+ mobilization from intracellular Ca2+ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca2+ accumulation by nonmitochondrial Ca2+ pools or the thapsigargin (TG)-sensitive Ca2+ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca2+ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca2+ . Therefore, NGF and dbcAMP induced different effects on Ca2+ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca2+ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca2+ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

BCL-2-related protein expression in apoptosis: oxidative stress versus serum deprivation in PC12 cells

Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H2O2 treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H2O2-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H2O2 but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H2O2 exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2''-(dimethylamino)ethyl]-1, 2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones with substitutions at the 6 and 7 positions (azonafides)

The p53 tumor suppressor gene encodes a phosphoprotein which when overexpressed can induce growth arrest at the G1 and G2/M phases of the cell cycle, promote differentiation and apoptosis. This paper demonstrates that p53 can associate with trk tyrosine kinase. Expression of a murine temperature-sensitive (ts) p53 mutant in PC12 cells overexpressing trk (a model system to analyse cellular differentiation and signal transduction induced by NGF) induces morphological changes in the absence of NGF stimulation at 32 degrees C but not at 37 degrees C. In cells differentiated by p53, trk, but not EGFr, was hyperphosphorylated on tyrosine. Furthermore trk was not phosphorylated when expressed in Saos-2 cells (human osteosarcoma cells that lack expression of both endogenous trk and p53) at either temperature. However, transfection of ts p53 into these cells induces trk phosphorylation at 32 degrees C in the absence of NGF stimulation. Association of trk and p53 can be detected in NIH3T3 and PC12 cells co-expressing trk and the ts p53 mutant, in NIH3T3 and PC12 cells transfected with trk alone, and in untransfected PC12 cells, showing that overexpressed and/or endogenous trk associates with endogenous, low levels of p53. These data suggest a novel function for p53 which involves the stimulation of signal transduction pathways (mediating morphological properties of cells), possibly through association with and hyperphosphorylation of trk.     

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.     

A role for MAPK/ERK in sympathetic neuron survival: protection against a p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside

The antimitotic nucleoside cytosine arabinoside (araC) causes apoptosis in postmitotic neurons for which two mechanisms have been suggested: (1) araC directly inhibits a trophic factor-maintained signaling pathway required for survival, effectively mimicking trophic factor withdrawal; and (2) araC induces apoptosis by a p53-dependent mechanism distinct from trophic factor withdrawal. In rat sympathetic neurons, we found that araC treatment for 12 hr induced approximately 25% apoptosis without affecting NGF-maintained signaling; there was neither reduction in the activity of mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) or protein kinase B/Akt, a kinase implicated in NGF-mediated survival, nor was there c-Jun N-terminal kinase (JNK) activation or c-Jun N-terminal phosphorylation, events implicated in apoptosis induced by NGF withdrawal. However, araC treatment, but not NGF-withdrawal, elevated expression of p53 protein before and during apoptosis. Additionally, araC-induced apoptosis was suppressed in sympathetic neurons from p53 null mice. Although MAPK/ERK activity is not necessary for NGF-induced survival, it protected against toxicity by araC, because inhibition of the MAPK pathway by PD98059 resulted in a significant increase in the rate of apoptosis induced by araC in the presence of NGF. Consistent with this finding, ciliary neurotrophic factor, which does not cause sustained activation of MAPK/ERK, did not protect against araC toxicity. Our data show that, in contrast to NGF deprivation, araC induces apoptosis via a p53-dependent, JNK-independent mechanism, against which MAPK/ERK plays a substantial protective role. Thus, NGF can suppress apoptotic mechanisms in addition to those caused by its own deprivation.     

N-terminal cysteines essential for Golgi sorting of B-50 (GAP-43) in PC12 cells

We have examined the subcellular distribution of the growth-associated protein B-50 (GAP-43) in pheochromocytoma (PC12) cells, using confocal microscopy. Proliferating PC12 cells contained very low levels of B-50 in the cytosol. Enhanced expression of B-50 in these cells, evoked by either nerve growth factor (NGF) treatment or transient transfection with rat B-50 cDNA, led to Golgi sorting and membrane targeting of the B-50 protein. Site directed mutagenesis of Cys3Cys4 to Ser3Gly4 in B-50 resulted in a cytosolic distribution. We conclude that Cys3, and Cys4 are essential for accumulation of B-50 both at the plasma membrane and in the Golgi apparatus of PC12 cells.     

Gonadotropin response to naloxone in the mare: effect of time of year and reproductive status

Effects of a novel dihydropyridine type of antihypertensive drug, cilnidipine, on the regulation of the catecholamine secretion closely linked to the intracellular Ca2+ were examined using nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells. By measuring catecholamine secretion with high-performance liquid chromatography coupled with an electrochemical detector, we showed that high K+ stimulation evoked dopamine release from PC12 cells both before and after NGF treatments. Cilnidipine depressed dopamine release both from NGF-treated and untreated PC12 cells in a concentration-dependent manner. In contrast, inhibition by nifedipine was markedly decreased in the differentiated PC12 cells. With intracellular Ca2+ concentration ([Ca2+]i) measurements using fura 2, the elevation of high K+-evoked [Ca2+]i was separated into nifedipine-sensitive and -resistant components. The nifedipine-resistant [Ca2+]i increase was also blocked by cilnidipine, as well as omega-conotoxin-GVIA. By the use of the conventional whole-cell patch-clamp technique, the compositions of the high-voltage-activated Ca2+ channel currents in the NGF-treated PC12 cells were divided into types: L-type, N-type, and residual current components. It was also estimated that cilnidipine at 1 and 3 micromol/L strongly blocked the N-type current without affecting the residual current. These results suggest that cilnidipine inhibits catecholamine secretion from differentiated PC12 cells by blocking Ca2+ influx through the N-type Ca2+ channel, in addition to its well-known action on the L-type Ca2+ channel.     

Mechanisms of radiation-induced chromosome aberrations

We have investigated the role of the low-affinity nerve growth factor (NGF) receptor p75NGFR in determining the death of neuronally differentiated PC12 cells after withdrawal of NGF. A range of high and low p75NGFR-expressing cells were obtained by a combination of fluorescence activated cell sorting (FACS) and stable transfection with a p75NGFR expression vector. Cells were readily differentiated to a neuronal phenotype irrespective of the level of p75NGFR expression. However, the rate and extent of neuronal death following NGF deprivation were extremely sensitive to the level of p75NGFR expression. The highest expressing cells died most rapidly. Cells selected for very low levels of p75NGFR expression exhibited resistance to NGF withdrawal, and remained as viable, differentiated neurons, with minimal cell death, for at least 5 days in the absence of NGF. Antisense oligonucleotides against p75NGFR were shown to down-regulate p75NGFR in PC12 cells and, further, to significantly enhance survival in the absence of NGF. These results consolidate and generalize our previous findings that p75NGFR induces cell death in postnatal sensory neurons in the absence of NGF. The ability to induce cell death in the absence of NGF appears to be a more general role of p75NGFR in differentiated neurons, and an important new paradigm for the mechanism of NGF-dependent survival.     

Physiological stress and nerve growth factor treatment regulate stress-activated protein kinase activity in PC12 cells

The stress-activated protein kinases (SAPKs) are differentially activated by a variety of cellular stressors in PC12 cells. SAPK activation has been linked to the induction of apoptotic cell death upon serum withdrawal from undifferentiated cells or following nerve growth factor (NGF) withdrawal of neuronally differentiated PC12 cells. However, withdrawal of trophic support from differentiated cells led to only a very modest elevation of SAPK activity and led us to investigate the basis of the relative insensitivity of these enzymes to stressors. NGF-stimulated differentiation of the cells resulted in the elevation of basal SAPK activity to levels four- to sevenfold greater than in untreated cells, which was correlated with an approximate fivefold increase in SAPK protein levels. Paradoxically, in NGF-differentiated PC12 cells, exposure to cellular stressors provoked a proportionately smaller stimulation of SAPK activity than that observed in naive cells, despite the presence of much higher levels of SAPK protein. The insensitivity of SAPK to activation by stressors was reflective of the activity of the SAPK activator SEK, whose activation was also diminished following NGF differentiation of the cells. The data demonstrate that SAPKs are subject to complex controls through both induction of SAPK expression and the regulation mediated by upstream elements within this pathway.     

Induction of neurofilament triplet proteins in PC12 cells by nerve growth factor

The localization of neurofilament triplet proteins in PC12 cells grown in the absence of (PC12-) or maintained in the presence of (PC12+) nerve growth factor (NGF) was studied using indirect immunofluorescence and monospecific, immunosorbent purified antibodies to 68,000 (P68), 150,000 (P150) and 200,000 (P200) dalton neurofilament proteins. The intensity of immunofluorescent staining of the triplet proteins was always greater in PC12+ compared with PC12-cells. Neuritic staining was seen in PC12+ cells with all 3 monospecific antibodies to neurofilament proteins. However, the perikaryal distribution of each of the neurofilament proteins differed in both PC12+ and PC12-cells. Monospecific antibodies to P68 protein yielded a 'ball-like' cytoplasmic staining pattern whereas monospecific antibodies by P150 protein stained in a stippled pattern. Monospecific antibodies to P200 on the other hand diffusely stained the perikaryal cytoplasm with very faint but detectable foci of 'ball-like' configurations and stippling. Electron microscopic study of PC12+ and PC12-cells revealed intermediate filaments in the cell bodies of both as well as in the processes of the former. 'Ball-like' clusters of such filaments were rarely seen. However, these filaments lacked the three-dimensional organization typical of intact neurofilaments. It is concluded that PC12 cells contain dissociated or incompletely assembled immunoreactive neurofilament triplet proteins and that these proteins can be induced by NGF. The PC12 cells are therefore an attractive model system not only for studies of neuronal differentiation but also for studies of neurofilament metabolism and disorders thereof.     

Plasmalopsychosine of human brain mimics the effect of nerve growth factor by activating its receptor kinase and mitogen-activated protein kinase in PC12 cells. Induction of neurite outgrowth and prevention of apoptosis

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.