Search

A short-term exposure of PC12 cells to tert-butylhydroperoxide, followed by recovery in fresh culture medium, causes cell death and the extent of this response progressively increases during the 120 min of post-treatment incubation. Morphological and biochemical analyses of these cells revealed that the mode of cell death was necrosis. Cell killing induced by the hydroperoxide seems to be in part mediated by peroxynitrite because the lethal response was markedly and similarly reduced by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methylester and by scavengers of nitric oxide or peroxynitrite. This peroxynitrite-dependent mechanism of cytotoxicity was blunted by antioxidants and inhibitors of mitochondrial permeability transition and the onset of cell death was preceded by mitochondrial depolarization and loss of cellular ATP. We conclude that tert-butylhydroperoxide promotes peroxynitrite-dependent PC12 cell necrosis causally linked to peroxidation of membrane lipids and mitochondrial permeability transition., (Copyright 2001 Wiley-Liss, Inc.)

In the present study, we investigated effects of estrogen on cell death induced by carboxy-terminal fragment of amyloid precursor protein (CT), a candidate causative substance in the pathogenesis of Alzheimer's disease. 17 beta-Estradiol attenuated CT-induced cell death in PC12 cells, whereas 17 alpha-estradiol, nonestrogenic stereoisomer, did not exert any significant protective effect on CT-induced cell death. These results suggest that protective effects of estrogen may be mediated by estrogen receptor (ER) in PC12 cells. To confirm the results, we determined the effects of tamoxifen, an estrogen receptor antagonist. Tamoxifen blocked the protective effects of 17 beta-estradiol, although it did not affect those of 17 alpha-estradiol. Overall, it might be thought that the protective effect of estradiol on CT-induced cell death is achieved by hormonal properties mediated through the estrogen receptor rather than the structural properties as a reducing agent., (Copyright 2001 Wiley-Liss, Inc.)

Ca2+ influx is one of the main causative events in hypoxic PC12 cell death, because an extracellular Ca2+ chelator, ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited and Ca2+ ionophore A23187 mimicked the hypoxic cell death. The hypoxic cell death was markedly prevented by a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) as well as a calpain inhibitor, calpeptin, as assessed by nuclear staining with Hoechst 33258 and lactate dehydrogenase release. The processing of procaspase-3 was inhibited by z-VAD-FMK, but not by calpeptin. In contrast, z-VAD-FMK failed to block the proteolytic cleavage of fodrin-alpha, a preferential substrate for calpain. On the other hand, degradation of actin and fodrin-alpha was prevented by calpeptin but not by z-VAD-FMK. In addition, not only protein kinase C (PKC)-alpha but also PKC-delta were cleaved to generate approximately 46 kDa fragments. The PKC fragmentation was inhibited by calpeptin but not by z-VAD-FMK. These findings suggest that the extracellular Ca2+ influx induced by hypoxic stress activates calpain, resulting in the degradation of cytoskeletal proteins and generation of PKC fragments almost independently of caspase activation. Therefore, calpain may play an important role in hypoxic PC12 cell death.

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indicator fluo-3) and immobilised using transparent polycarbonate membrane filters with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2+ concentration in the PC12 cells, followed by a delayed secondary Ca2+ response in simultaneously monitored nearby HEL cells. There was a lag period of about 20 s between the responses of the two cell types. Voltage-gated Ca2+ channels in PC12 cells were inhibited by the P/Q-type (omega-conotoxin MVIIC, omega-agatoxin IVA), N-type (omega-conotoxin GVIA) and L-type channel blockers (nifedipine) as determined using fura-2 or whole-cell patch-clamp recordings. The communication between the cell types on the other hand was sensitive to P/Q- and N-type but not to L-type channel blockers. This suggests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the release of neurotransmitters acting on HEL cells. Theoretically, the procedure employed should be sensitive enough to detect single exocytotic events. Our results demonstrate that a random distribution between effector and target cells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.

Beta-amyloid (Abeta) peptides, the major component of senile plaques in brains of patients with Alzheimer's disease (AD), were found in the low pH organelles (i.e. endosome/lysosome) of cultured neuronal cells. Since acidic pH values have been shown to promote the self-assembly of Abetas, which seems to be a prerequisite for their neuropathogenicity, elucidating the aggregation behavior of Abetas in acidic environments and their subsequent effects on neuronal cells may be crucial for understanding the neurodegenerative process of AD. In this study, the extent and rate of aggregation of Abeta(1-42) peptides at pH values of 5.8 and 7.4, as well as the structure and neurotoxic effects of these aggregates, were examined. We showed that Abeta(1-42) peptides formed large and complex fibrils much more efficiently at acidic rather than neutral pH. Furthermore, only the pH 5.8 Abeta aggregates induced significant apoptotic death of PC12 cells, as indicated by a decrease in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) reduction and an increase in phosphatidylserine externalization. Taken together, our results suggest that the Abetas present in the acidic organelles may form neurotoxic fibrils more easily than those in the neutral cellular compartments.

TRK-T1 oncogene is generated by the rearrangement of the NGF receptor TrkA with TPR. This gives rise to the constitutive tyrosine autophosphorylation and activation of the kinase. To study TRK-T1 oncogenic signaling and compare it to that induced by the genuine receptor TrkA, we investigated the involvement of IRS-1, a docking protein implicated in mitogenic signaling induced by several growth factors, in TRK-T1 and TrkA signaling. Here, we show that IRS-1 and IRS-2 are phosphorylated on tyrosine in presence of both TRK-T1 and the activated TrkA receptor. These tyrosine phosphorylations lead to IRS-1- and IRS-2-induced recruitment of p85PI3K, SHP-2, and Grb2 and increase in PI 3-kinase activity associated with IRS-1. Furthermore, we found that TRK-T1 is able to activate c-fos serum responsive element in cooperation with IRS-1 and IRS-2. We observed that TRK-T1 stimulates DNA synthesis in wild-type fibroblasts but not in IRS-1(-/-) mouse embryo fibroblasts. Yeast two-hybrid system experiments showed the occurrence of direct interaction between TRK and IRS molecules, which suggests involvement of different modes of interactions. On the whole, our results suggest that IRS-1 and IRS-2 could be substrates of TRK-T1 and TrkA, and hence could participate in their signal generation.

Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation., (Copyright 2001 Wiley-Liss, Inc.)

A number of studies indicate that free radicals are involved in the neurodegeneration in Alzheimer's disease (AD). The present study was mainly conducted to examine the effect of Huperzine B on H(2)O(2) induced toxicity in rat pheochromocytoma line PC12 by measuring cell lesion, level of lipid peroxidation and antioxidant enzyme activities. Following a 30 min exposure of the cells to H(2)O(2) (150 microM), a marked decrease in cell survival, activities of glutathione peroxidase and catalase as well as increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with huperzine B (10-100 microM) prior to H(2)O(2) exposure significantly elevated the cell survival, antioxidant enzyme activities and decreased the level of MDA. The above-mentioned neuroprotective effects are also observed with tacrine (1 microM), donepezil (10 microM) and galanthamine (10 microM), suggesting that the neuroprotective effects of cholinesterase inhibitor might partly contribute to the clinical efficacy in AD treatment.

HES-1 is a Hairy-related basic helix-loop-helix protein with three evolutionarily conserved regions known to define its function as a transcription repressor. The basic region, helix-loop-helix domain, and WRPW motif have been characterized for their molecular function in DNA binding, dimer formation, and corepressor recruitment, respectively. In contrast, the function conferred by a fourth conserved region, the helix 3-helix 4 (H-3/4) domain, is not known. To better understand H-3/4 domain function, we expressed HES-1 variants under tetracycline-inducible control in PC12 cells. As expected, the induced expression of moderate levels of wild-type HES-1 in PC12 cells strongly inhibited nerve growth factor-induced differentiation. This repression was dependent on the H-3/4 domain. Unexpectedly, expression of HES-1 also arrested cell growth, an effect that could be reversed upon down regulation of HES-1. Concomitant with growth arrest, there was a strong reduction in bromodeoxyuridine incorporation and PCNA protein levels, although not in cyclin D1 expression. Expression of a HES-1 protein carrying the H-3/4 domain, but not the WRPW domain, still partially inhibited both proliferation and differentiation. Transcription assays in PC12 cells directly demonstrated that the H-3/4 domain can mediate DNA-binding-dependent transcription repression, even in the absence of corepressor recruitment by the WRPW motif. HES-1 expression strongly repressed transcription of the p21(cip1) promoter, a cyclin-cyclin-dependent kinase inhibitor up regulated during NGF-induced differentiation, and the H-3/4 domain is necessary for this repression. Thus, the H-3/4 domain of HES-1 contributes to transcription repression independently of WRPW function, inhibits neurite formation, and facilitates two distinct and previously uncharacterized roles for HES-1: the inhibition of cell proliferation and the direct transcriptional repression of the NGF-induced gene, p21.

The effects of huperzine A (HupA), a novel acetylcholinesterase inhibitor, on Abeta(25-35)-induced cell lesion, level of lipid peroxidation, antioxidant enzyme activities were investigated in the rat pheochromocytoma line PC12. Following a 48 h exposure of the cells to Abeta(25-35), a significant reduction in cell survival and activities of glutathione peroxidase (GSH-Px) and catalase (CAT), as well as increased production of malondialdehyde (MDA) and superoxide dismutase (SOD) were observed. Preincubation of the cells with HupA prior to Abeta(25-35) exposure elevated the cell survival and GSH-Px and CAT activities, and decreased the level of MDA and SOD activity. The results indicate that HupA has protective effects against Abeta-induced cell toxicity, which might be beneficial for the treatment of Alzheimer's disease.

Proteins are secreted from animal cells by either a constitutive or a regulated pathway. When cDNA of nerve growth factor (NGF) was introduced into PC12 cells, these cells produced and secreted active NGF, where NGF was secreted not only in constitutive but also in activity-dependent regulated way according to the results of pulse-chase and ELISA studies. The regulated secretion was caused by depolarization, cyclic AMP analogue, or beta-adrenergic agonist but not by glutamate or carbachol. Because these transfected cells differentiated into a morphology indistinguishable from that incubated with NGF protein, we next compared the secretion pathways of NGF from PC12 cells at different stages of the differentiation. NGF was secreted in both constitutive and regulated way at 2 and 7 days after the transfection of NGF-cDNA, but the constitutive secretion of NGF from the more differentiated cells of Day 7 was decreased and mature NGF tended to accumulate in the cells. These results indicate that the neurotrophin secretion mechanism is intimately regulated in the course of the differentiation of PC12 cells. Such a change in the protein secretion pathway might have an profound role in the development of neurons., (Copyright 2000 Wiley-Liss, Inc.)

Neu differentiation factor (NDF; also known as neuregulin) induces a pleiotropic cellular response that is cell type-dependent. NDF and its receptor ErbB-4 are highly expressed in neurons, implying important roles in neuronal cell functions. In the present study we demonstrate that ErbB-4 receptors expressed in PC12 cells mediate NDF-induced signals and neurite outgrowth that are indistinguishable from those mediated by the nerve growth factor-activated Trk receptors. In PC12-ErbB-4 cells but not in PC12 cells, NDF induced an initial weak mitogenic signal and subsequently neurite outgrowth. The NDF-induced differentiation in PC12-ErbB-4 cells was mimicked by the pan-ErbB ligand betacellulin but not by other epidermal growth factor-like ligands. Thus, NDF and betacellulin mediate similar activities through the ErbB-4 receptor. Indeed, only these ligands induced strong phosphorylation of the ErbB-4 receptors. Neurite outgrowth induced by NDF in PC12-ErbB-4 cells was accompanied by sustained activation of mitogen-activated protein kinase (MAPK) and induction of the neural differentiation marker GAP-43. Inhibition of the MAPK kinase MEK or of protein kinase C (PKC) blocked NDF-induced differentiation, whereas elevation of cyclic AMP levels enhanced the response. Taken together, these results indicate that neurite outgrowth induced by ErbB-4 in PC12 cells requires MAPK and PKC signaling networks.

The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress.

It is well known that neuronal differentiation is strongly dependent on the intracellular level of free calcium ions ([Ca2+]i). In the present study the morphological and intracellular free calcium concentration changes were compared on PC12 pheochromocytoma cells cultured in control conditions and in a medium with high KCl level. Culturing PC12 cells in a medium with 20-30 mM KCl deprived of nerve growth factor supported cell proliferation and rapid growth of small neurite-like processes. However, their lengths did not increase with prolongation of the time of culturing. During culturing with 40 mM KCl the growth of these processes became blocked; the cells stopped proliferating and showed signs of degeneration. Measurements of [Ca2+]i level during the first days of PC12 cells culturing in a hyperpotassium medium indicate that such changes in this level could be an important factor in the induction of the observed morphological alterations; however, other effects induced by membrane depolarization may also be responsible for them.

Protein 4.1N is a neuronal selective isoform of the erythrocyte membrane cytoskeleton protein 4.1R. In the present study, we demonstrate an interaction between 4.1N and nuclear mitotic apparatus protein (NuMA), a nuclear protein required for mitosis. The binding involves the C-terminal domain of 4.1N. In PC12 cells treatment with nerve growth factor (NGF) elicits translocation of 4. 1N to the nucleus and promotes its association with NuMA. Specific targeting of 4.1N to the nucleus arrests PC12 cells at the G1 phase and produces an aberrant nuclear morphology. Inhibition of 4.1N nuclear translocation prevents the NGF-mediated arrest of cell division, which can be reversed by overexpression of 4.1N. Thus, nuclear 4.1N appears to mediate the antiproliferative actions of NGF by antagonizing the role of NuMA in mitosis.

Background: Riluzole is known to be an inhibitor of glutamatergic neurotransmission. Transmitter release from nerve terminals can be regulated by the activity of large-conductance Ca(2+)-activated K+(BKCa) channels., Methods: The ionic mechanism of actions of riluzole was investigated in neuroendocrine (GH3 and PC12 cells), using the whole-cell patch-clamp and inside-out excised patch configurations., Results: In GH3 cells, riluzole at 0.3-100 mumol/L increased the amplitude of Ca(2+)-activated K+ current (IK(Ca)) in a concentration-dependent manner with a half maximal concentration of 5 mumol/L. The riluzole-induced increase in outward current was not be suppressed by glibenclamide (10 mumol/L) or apamin (200 nmol/L). However, iberiotoxin (200 nmol/L) or tetrandrine (10 mumol/L) can effectively suppress riluzole-induced IK(Ca). Under inside-out patch recording mode, riluzole (10 mumol/L) applied intracellularly can increase the opening probability of large-conductance Ca(2+)-activated K+(BKCa) channels, but did not affect their single-channel conductance. The riluzole-induced change in the kinetic behavior of BKCa channels is due to an increase in mean open time and a decrease in mean closed time. Riluzole caused a left shift in the midpoint for voltage-dependent opening. Riluzole-stimulated activity of BKCa is independent on internal Ca2+. Riluzole (30 mumol/L) did not affect the amplitude of voltage-dependent K+ current, but it produced a slight reduction of L-type voltage-dependent Ca2+ current. Under current clamp mode, riluzole (10 mumol/L) decreased the firing rate of action potentials induced by thyrotropin releasing hormone (10 mumol/L) in GH3 cells. In rat pheochromocytoma PC12 cells, riluzole also increased the activity of BKCa channels without altering their channel conductance., Conclusion: This study shows that riluzole can stimulate the activity of BKCa channel in neuroendocrine cells.

Neuronal apoptosis in rat pheochromocytoma PC12 cells, which was confirmed by TUNEL (terminal transferase-mediated dUTP-biotin nick end-labeling) staining and detection of chromatin condensation, appeared within 8 h after nerve growth factor (NGF) deprivation. Prostaglandin (PG) E1 (10(-7)-10(6) M) reduced the incidence of apoptotic cell death in PC12 cells. The genes encoding PG transporter specific to prostaglandins such as PGE2 or PGF2alpha were expressed in the cell lines as shown by RT-PCR. Bromcresol green, an inhibitor of PG transporter, reversed the antiapoptotic effect of PGE1. Moreover, treatment of PC12 cells with an antisense oligonucleotide corresponding to PG transporter cDNA also blocked the inhibitory effects of PGE1 on apoptotic cell death. In addition, PGE1 counteracted the increased activities of stress-activated protein kinase/cJun N-terminal kinase within 1-2 h after NGF deprivation in PC12 cells. These results indicated that the antiapoptotic effect of PGE1 in NGF-deprived PC12 cells was achieved by inhibitory signals following uptake into neurons through the PG transporter.

The rat pheochromocytoma PC12 cells have been employed as a useful model to study neuronal differentiation. To gain insight into the molecular events involved in extension of neurites, the differential expression of phospholipase D (PLD) isozymes were examined in cyclic AMP- or nerve growth factor (NGF)-treated PC12 cells. When the cells were incubated with dibutyryl cyclic AMP (dbcAMP), almost all the cells displayed prominent neurite outgrowth at 24 h. The neurites developed in NGF-treated cells at 24 h were shorter than those in the cells treated with dbcAMP. However, most of the NGF-treated cells extended long neurites at day 5. The rPLD1b mRNA increased within 6 h following dbcAMP treatment and maintained a high level up to 24 h. In contrast, the levels of rPLD1a and rPLD2 mRNAs were rather consistent throughout the time course examined. However, when the cells were treated with NGF, rPLD1a and rPLD1b mRNAs, but not rPLD2 mRNA, increased within 2 days and remained elevated up to 5 days. These results suggest the possible implication of PLD1 in PC12 cell differentiation.

The mechanism of proadrenomedullin N-terminal 20 peptide (PAMP)-induced inhibition of catecholamine release from adrenergic nerve was investigated in nerve growth factor-treated PC12 cells that have differentiated characteristics somewhat similar to noradrenergic neurons. The effect of PAMP on the excitability of these cells was investigated with the use of perforated whole-cell clamp. PAMP hyperpolarized the membrane by increasing a K+ conductance in a dose-dependent manner. The current-voltage relationship (I-V) relationship of the PAMP-induced K+ conductance exhibited inward-going rectification. The activation was abolished by microinjecting GDPbetaS into the cells or pretreating the cells with pertussis toxin. These results indicate that a pertussis toxin-sensitive G protein is involved in the signal transduction. The PAMP-induced activation of the K+ conductance was attenuated by microinjecting antibody against the carboxyl terminus of Galphai3, but it was not influenced by microinjecting antibody against the common carboxyl termini of Galphai1 and Galphai2, which indicated that the G protein coupling the PAMP receptor to the inwardly rectifying K+ current is Galphai3. The PAMP-induced hyperpolarization may inhibit the catecholamine release from the neurons by attenuating the action potential frequency.

Understanding the pathogenesis of Alzheimer's disease is of widespread interest because it is an increasingly prevalent disorder that is progressive, fatal, and currently untreatable. The dementia of Alzheimer's disease is caused by neuronal cell death. We demonstrate for the first time that blood vessels isolated from the brains of Alzheimer's disease patients can directly kill neurons in vitro. Either direct co-culture of Alzheimer's disease microvessels with neurons or incubation of cultured neurons with conditioned medium from microvessels results in neuronal cell death. In contrast, vessels from elderly nondemented donors are significantly (P<0.001) less lethal and brain vessels from younger donors are not neurotoxic. Neuronal killing by either direct co-culture with Alzheimer's disease microvessels or conditioned medium is dose- and time-dependent. Neuronal death can occur by either apoptotic or necrotic mechanisms. The microvessel factor is neurospecific, killing primary cortical neurons, cerebellar granule neurons, and differentiated PC-12 cells, but not non-neuronal cell types or undifferentiated PC-12 cells. Appearance of the neurotoxic factor is decreased by blocking microvessel protein synthesis with cycloheximide. The neurotoxic factor is soluble and likely a protein, because its activity is heat labile and trypsin sensitive. These findings implicate a novel mechanism of vascular-mediated neuronal cell death in Alzheimer's disease.

Previously, we showed that 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) is an effective inducer of melanogenesis in cultured cells and guinea-pig skin [Brown et al. (1998) J. Invest. Dermatol., 110:428-437]. This study shows that 2,3-cis/exo-pinanediol (2,3-cs/ex-PinD) is a more effective inducer of melanogenesis than 5-NBene-2,2-DM in S91 mouse melanoma cells. Furthermore, 2,3-cs/ex-PinD appears to penetrate guinea-pig skin better than 5-NBene-2,2-DM and to induce higher levels of pigmentation. Both 5-NBene-2,2-DM and 2,3-cs/ex-PinD induce synthesis of nitric oxide (NO) in S91 cells, and the melanogenic activity of both compounds is reduced by inhibitors of the NO/cyclic guanosine monophosphate (cGMP)/protein kinase(PK) G signaling pathway, but not by inhibitors of the PKC or PKA pathways. Thus, these bicyclic monoterpene diols appear to induce melanogenesis by the same pathway in S91 cells as that shown previously for ultraviolet radiation in melanocytes (Romero-Graillet et al. (1996) J. Biol. Chem., 271:28052-28056). These compounds also induce NO synthesis, neurite outgrowth, and tyrosine hydroxylase activity in PC12 pheochromocytoma cells. Neurite outgrowth in PC12 cells is blocked by the guanylate cyclase inhibitor, LY83583 (6-anilino-2,8-quinolinequinone), indicating that, similar to S91 cells, the induction of morphological differentiation of PC12 cells by bicyclic monoterpene diols is regulated by a cGMP-dependent pathway.

Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.

Translational rates, and activities and levels of initiation factors 2 and 2B were assessed in rat pheochromocytoma cells upon nerve growth factor (NGF) treatment. Two or 5 days of exposure to NGF caused significant quantitative increases in protein synthesis rate that are deemed necessary for neuronal differentiation. Changes in initiation factor 2 activity, as measured by its capacity to form a ternary complex, occur parallel to the observed changes in protein synthesis. Nevertheless, neither the intracellular levels of the initiation factor 2 nor the degree of phosphorylation of its alpha subunit can justify this increased activity. Interestingly, initiation factor 2B activity increases parallel to the neurite outgrowth, being significantly higher after 5 days of exposure to NGF, and could be responsible for the elevated rate of protein synthesis. No significant changes in the levels of eukaryotic initiation factor 2B, as determined with two different antibodies against the gamma and epsilon subunits of the factor, were observed, implying that the increased activity should be regulated by factors other than its cellular concentration. Our results support the hypothesis that initiation factor 2B may play a role in the biochemical events controlling the differentiative growth factor-induced signaling pathway in these cells.

Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) in chick embryos results in a 30-40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp-8-pCPT-cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp-8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.

Glial cells play an important role in maintaining neural function. In the present study, we examined the effects of a factor derived from human astrocytoma cells (1321N1) on differentiation of rat pheochromocytoma cells (PC-12). The conditioned medium which had been used for culture of 1321N1 cells caused the differentiation of PC-12 cells, suggesting that 1321N1 cells release a neurotrophic factor. The factor was apparently distinct from well-known neurotrophic factors, such as nerve growth factor (NGF), since it was resistant to boiling and trypsin treatment. The molecular size of the factor was assumed to be below 1000 through dialysis and ultrafiltration experiments. Furthermore, PC-12 cells were differentiated synergistically by the combined addition of NGF and the conditioned medium of 1321N1 cells. Partially purified fraction of the factor by Sephadex G-15 gel filtration column caused the prolonged activation of mitogen-activated protein kinase (MAPK). The differentiation of PC-12 cells induced by the fraction or NGF disappeared after the treatment with PD98059, a specific inhibitor of MAPK kinase (MEK), suggesting the involvement of MAPK in the differentiation. These results suggest that the new low-molecular factor derived from glial cells causes differentiation of PC-12 cells mediated through an activation of MAPK., (Copyright 1998 Elsevier Science B.V.)

The two MAP kinases JNK and ERK direct distinct cellular activities even though they share a number of common substrates, including several transcription factors. Here we have compared JNK and ERK signalling during PC12 cell differentiation and investigated how activation of c-Jun by the MAPKs contributes to this cellular response. Exposure to nerve growth factor, or expression of constitutively active MEK1-two treatments which cause differentiation of PC12 cells into a neuronal phenotype-result in activation of ERK-type MAP kinases and phosphorylation of c-Jun on several sites including Ser63 and Ser73. Constitutively activated c-Jun, which mimics the MAPK-phosphorylated form of the protein, can induce neuronal differentiation of PC12 cells independently of upstream signals. Conversely, expression of dominant-negative c-JunbZIP prevents neurite outgrowth induced by activated MEK1. Activation of MEKK1, which stimulates the JNK pathway, is not sufficient for PC12 cell differentiation but can induce apoptosis. However, neurite outgrowth is triggered when c-Jun is co-expressed with activated MEKK1 or SEK1. Consistently, MEK-induced ERK activation in PC12 cells induces c-Jun expression, while JNK signalling does not. Therefore, dual input of expression and phosphorylation of c-Jun provided by the ERK pathway is required to direct neuronal differentiation in PC12 cells.

Characteristics of Alzheimer's disease include loss of brain neurons associated with the deposit of beta-amyloid protein (A beta) which is thought to be toxic to neurons possibly via induction of intracellular calcium and generation of free radicals. On this basis, we have determined the effect of ascorbic acid on the cell death and calcium increase induced by A beta in PC12 cells. We found that ascorbic acid completely abolished A beta-induced calcium increase and cell death in PC12 cells, indicating that calcium elevation and cell death are associated phenomena induced by A beta that can be rescued by antioxidants. These results are important to understand the mechanisms by which A beta is toxic to neurons and suggest that antioxidants may be part of future treatments for Alzheimer's disease.

Neuron-like cells derived from a rat pheochromocytoma cell line (PC12) and differentiated with nerve growth factor produce a paired helical filament (PHF)-like antigen when they are subjected to heat shock (Wallace et al.: Mol Brain Res 19:149-155, 1993). It accumulates in a localized region of the perinuclear cytoplasm and reacts with monoclonal antitau antibodies, which identify epitopes in the N- and C-terminal halves and the microtubule-binding domain of tau protein. The observed profile of immunoreactivity suggests the presence of full-length and C-terminally truncated tau in a region of perinuclear cytoplasm in which no structurally intact PHFs could be demonstrated by conventional transmission electron microscopy. The accumulated tau protein colocalized with antibodies raised against mitochondrial outer membrane proteins and was associated with the presence of numerous mitochondrial profiles that were demonstrated with electron microscopy. Because differentiated PC12 cells pretreated with colcemid or Taxol prior to heat shock fail to exhibit perinuclear PHF-like immunoreactivity, the reported response to heat shock appears to require an intact system of intracellular microtubules. This PC12 system provides a model in which the metabolic and molecular biological underpinnings of neuronal degeneration in Alzheimer's disease can be manipulated. The system may eventually be applicable to the development of pharmaceutical agents that interfere with formation and/or degeneration of PHF-tau in Alzheimer's disease.

Reportedly, the generation of nitric oxide (NO) may lead to iron mobilization from ferritin disrupting intracellular iron homeostasis and increasing levels of reactive oxygen species. In the present study, we evaluated the role of endogenous iron in NO-induced apoptosis in PC12 cells. Apoptosis was tested by flow cytometry, fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine 5'-triphosphate nick end labeling (TUNEL) technique. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When incubated with 0.5-0.75 mM sodium nitroprusside (SNP, a chemical NO donor), PC12 cells were shown to undergo apoptosis. In addition, SNP induced a time-dependent decrease in cell viability. Since deferoxamine (0.05-0.1 mM), a powerful iron chelator, inhibited both SNP-induced apoptosis and the decrease in cell viability, we suggest that these NO effects may be dependent upon iron mobilization within the cell.

Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto)chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.

We previously demonstrated, using rat PC-12 pheochromocytoma cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF), that neuropeptide Y (NPY) inhibits catecholamine synthesis as well as release. Inquiry into the mechanisms of these inhibitions implicated distinct pathways involving reduction of Ca2+ influx through voltage-activated Ca2+ channels. In the present investigation the effects of NPY on whole cell Ba2+ currents were examined to obtain direct evidence supporting the mechanisms suggested by those studies. NPY was found to inhibit the voltage-activated Ba2+ current in NGF-differentiated PC-12 cells in a reversible fashion with an EC50 of 13 nM. This inhibition was pertussis toxin sensitive and resulted from NPY modulation of L- and N-type Ca2+ channels. The inhibition of L-type channels was not seen with < 1 nM free intracellular Ca2+ or when protein kinase C (PKC) was inhibited by chelerythrine or PKC-(19-31). Furthermore, the effect of NPY on L-type channels was mimicked by the PKC activator phorbol 12-myristate 13-acetate. These studies demonstrate that, in addition to inhibition of N-type Ca2+ channels, in NGF-differentiated PC-12 cells NPY inhibits L-type Ca2+ channels via an intracellular Ca(2+)- and PKC-dependent pathway.

The glycation reaction (Maillard reaction) plays a major role in diabetic complications, since some reaction intermediates are responsible for the modification and cross-linking of long-lived proteins, resulting, in turn, in a deterioration of normal cell function. The reaction intermediates include methylglyoxal (MG) and 3-deoxyglucosone (3-DG), both of which are cytotoxic dicarbonyl compounds and are elevated during hyperglycemia. Aldehyde reductase (ALR) catalyzes the reduction of both compounds. To examine the intracellular role of ALR in the diabetic complications of neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells, which normally express a low level of ALR. Western blot analysis showed that ALR protein in the ALR gene-transfected cells was more than twice as much as in the control cells. In the parental cells, cytotoxicity, including apoptotic cell death, which was determined by fluorescent microscopy using the fluorescent DNA binding dye Hoechst 33258, was observed at 100 microM MG. In the ALR gene-transfected cells, the cytotoxicity of both MG and 3-DG and apoptotic cell death were decreased. This suggests that intracellular ALR protects neural cells from the cytotoxicity of 3-DG or MG, and that neural cells, which normally express a low level of ALR, might be susceptible to diabetic complications caused by intermediate products of the Maillard reaction, such as 3-DG and MG.

Tau is a microtubule-associated protein whose promoter is activated during the first phase of nerve growth factor-induced PC12 cell differentiation, whereas levels of its mRNA are accumulating throughout differentiation. In this study, we have followed the signal transduction cascades regulating tau induction. Using dominant negative Ras-expressing PC12 cells, we show that ras regulates tau expression during the first phase of PC12 cell differentiation. The ERK and JNK cascades, which are downstream of Ras, have opposing effects on tau promoter activity: ERK induces tau promoter activity, JNK inhibits it. Tau promoter activity in PC12 cells is correlated with a short-term activation of ERK, which declines after a few hours and is followed by an activation of the inhibitory JNK cascade 76 h later. These observations suggest that the induction and inhibition of tau promoter are mediated by alternate ERK and JNK activities, which may underlie a mechanism to turn on and off genes during PC12 cell differentiation.

Rat pheochromocytoma PC12 cells differentiate into nonreplicating neuronal cells with neurite extensions in response to nerve growth factor (NGF). To gain better understanding of the regulation of stress responses in neuronal cells, we examined the induction of HSP70, HSP70 mRNA, and heat shock factor 1 (HSF1) DNA-binding activity following treatment by heat shock or with sodium arsenite or amino acid analogue in PC12 cells treated with or without NGF. The induction of HSP70 and HSP70 mRNA following these stresses was diminished in the differentiated PC12 cells compared to the undifferentiated cells, whereas the HSF1 DNA-binding activity was enhanced in the differentiated PC12 cells. This phenomenon was characteristic of the differentiated neuronal cells rather than growth-arrested cells. Thus, neuronal cells appear to show an altered stress response depending on their differentiation state, and the diminished HSP70 expression in the differentiated neuronal cells may explain the sensitivity of neuronal cells to pathophysiological stressors.

Epidermal Langerhans cells are frequently anatomically associated with calcitonin gene-related peptide-containing nerves. Furthermore, calcitonin gene-related peptide inhibits Langerhans cells antigen-presenting function in several assays. Studies were performed to further explore the hypothesis that Langerhans cells and nerves have a functional relationship. To examine whether Langerhans cells may produce factors that influence nerve cell differentiation, we utilized the Langerhans cell-like cell line XS52 as a surrogate for Langerhans cells and compared it with Langerhans cells enriched to 90%. Supernatants conditioned by lipopolysaccharide-stimulated XS52 cells were able to induce the differentiation of the pheochromocytoma line PC12 into sympathetic neuron-like cells. This was also the case with enriched Langerhans cells stimulated by lipopolysaccharide. Pretreatment of conditioned supernatants with specific neutralizing anti-sera indicated that most of the differentiation-inducing activity was due to interleukin-6 and a small amount was due to nerve growth factor and basic fibroblast growth factor. By reverse transcriptase polymerase chain reaction, three clones of the XS52 cell line, XS52-4D, XS52-11D, and XS52-8B, were found to express mRNA for interleukin-6 and expression was markedly augmented by lipopolysaccharide. mRNA for nerve growth factor and basic fibroblast growth factor was detected in XS52-4D and XS52-11D, but not in XS52-8B. The expression of these neurotrophic factors by enriched Langerhans cells was quite similar to that of XS52-4D. In order to examine whether Langerhans cells may express receptors for nerve-derived peptides, reverse transcriptase polymerase chain reaction was employed to look for pituitary adenylate cyclase activating polypeptide type I, type II, and type III, and gastrin-releasing peptide receptors. All clones examined, as well as enriched Langerhans cells, expressed pituitary adenylate cyclase activating polypeptide type II and type III, and gastrin-releasing peptide receptors. These results suggest bi-directional signalling between Langerhans cells and nerves; nerve cells may regulate Langerhans cell function by elaboration of certain neuropeptides whereas Langerhans cells may promote the differentation of nerves by elaboration of interleukin-6 and, possibly, other factors.

The effects of cholesterol oxides on regular and neuronal PC12 cells were examined. Among the cholesterol oxides tested, 7-beta-, 22(R)- and 25-OH-cholesterol were very toxic to regular PC12 cells such that approximately 80% of cells were killed after a 3 day incubation with 20 micrograms/ml (50 microM) of these agents. Cholesterol oxides caused cytotoxicity in neuronal PC12 cells at concentrations higher than those observed for regular PC12 cells. The nerve growth factor (NGF)-induced neurite outgrowth and nitric oxide synthase expression appeared normal in PC12 cells treated with a sublethal dose of 25-OH-cholesterol. Neurite regeneration from established neuronal PC12 cells was not inhibited by the presence of a toxic dose of 25-OH-cholesterol. Given the fact that 25-OH-cholesterol is a potent inhibitor of cholesterol synthesis pathway, these results suggest that the de novo synthesis of cholesterol may not be required for NGF-induced neurite outgrowth in PC12 cells.

Peroxide formation has been implicated in impairment of motor function by cyanide which occurs in both animals and man. The present study employs the neuronal model, rat pheochromocytoma (PC12) cells to evaluate peroxidation as a toxic mechanism of cyanide. Confocal imaging shows that peroxides form within a few seconds in cell cytoplasm after cyanide exposure and continue to accumulate over a period of several minutes. Peroxide generation by cyanide is decreased to about 50% by phospholipase A2 inhibitors indicating involvement of arachidonic acid in the oxidative process. Also antioxidant defense enzymes (CuZn superoxide dismutase and especially catalase) in PC12 cells are inhibited by cyanide. It appears that peroxide accumulation after cyanide treatment involves both inhibition of breakdown and increased production. Furthermore, both peroxide accumulation and cell death induced by cyanide in PC12 cells are blocked by an antioxidant (ascorbate). These data support the hypothesis that the cytotoxic action of cyanide is related in part to an oxidative process.

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.

A clonal cell line stably expressing trkB (TrkB/ PC12) was established from rat pheochromocytoma PC12 cells. Brain-derived neurotrophic factor (BDNF), as well as nerve growth factor (NGF), stimulates neurite outgrowth in TrkB/PC12 cells. However, the morphology of BDNF-differentiated cells was clearly different from NGF-differentiated cells. BDNF treatment brought about longer and thicker neurites and induced a flattened soma and an increase in somatic size. This is not explained enough by the quantitative difference in the strength between TrkA and TrkB stimulation, because the level of BDNF-stimulated tyrosine phosphorylation of TrkB was similar to that of TrkA stimulated with NGF in PC12/TrkB cells. There was no difference in major tyrosine phosphorylated proteins induced by NGF and BDNF. Signal proteins such as phosphatidylinositol 3-kinase, phospholipase C-gamma 1, Shc, and mitogen-activated protein kinase seem to be involved in both TrkA- and TrkB-mediated signaling pathways. However, a tyrosine-phosphorylated 38-kDa protein (pp38) was detected in anti-pan-Trk immunoprecipitation only after NGF stimulation. Immunoprecipitation using three distinct anti-pan-Trk antibodies suggests that pp38 is not a fragment of TrkA. These data indicate that TrkA has a unique signal transduction pathway that is not stimulated through TrkB in TrkB/PC12 cells and suggest distinct functions among neurotrophin receptors.

Studies from several laboratories have generated evidence suggesting that oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD). The finding that the amyloid beta protein (Abeta) has neurotoxic properties and that such effects are, in part, mediated by free radicals has provided insights into mechanisms of cell death in AD and an avenue to explore new therapeutic approaches. In this study we demonstrate that melatonin, a pineal hormone with recently established antioxidant properties, is remarkably effective in preventing death of cultured neuroblastoma cells as well as oxidative damage and intracellular Ca2+ increases induced by a cytotoxic fragment of Abeta. The effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including cell viability studies by confocal laser microscopy, electron microscopy, and measurements of intracellular calcium levels. The importance of this finding is that, in contrast to conventional antioxidants, melatonin has a proposed physiological role in the aging process. Secretion levels of this hormone are decreased in aging and more severely reduced in AD. The reported phenomenon may be of therapeutic relevance in AD.

Microtubules are stiff polymers that extend throughout the cytoplasm of eukaryotic cells and regulate the location of intracellular compartments. Microtubules are formed by the polymerization of tubulin molecules, each of which is a heterodimer consisting of two closely related globular polypeptides, alpha-tubulin and beta tubulin. Many of the microtubule arrays are labile and are turning over rapidly; they depolymerize and repolimerize continually in living cells. One of the most striking examples is the mitotic spindle, which forms after the cytoplasmic microtubules disassemble at the onset of mitosis. The mitotic spindle is the target of a variety of agents that act by interfering with the exchange of tubulin subunits between the microtubules and the free tubulin pool; some of these agents can be used as antimitotic or anticancer drugs. We recently have developed a simple and sensitive method of screening for tubulin inhibitors, to which we have successfully applied the nerve growth factor/PC12 cell system. In this report, we describe the details of this method for screening tubulin inhibitors, by which we have analyzed several well-known and new compounds a model experiments.

To investigate the regulation of norepinephrine transporters (NETs) in vitro, we measured the binding of the NET-selective ligand [3H]nisoxetine in homogenates of PC12 cells after exposure of intact cells to the NET inhibitor desipramine (DMI). A 3-day exposure of PC12 cells to DMI robustly reduced the Bmax, but not the KD, of [3H]nisoxetine binding to NETs. Reduction of the binding of [3H]nisoxetine was dependent on both the concentration of DMI and the time of exposure to DMI. Reduction of [3H]nisoxetine binding to NETs produced by a 1-day exposure to DMI reverted to preexposure levels 48 h after cessation of DMI exposure. Similar down-regulation of NETs was found when PC12 cells were exposed to another NET-selective drug, nisoxetine, which is structurally unrelated to DMI. In contrast, exposure of cells to the serotonin transporter-selective drug citalopram, or the NET substrate norepinephrine, had no effects on the binding of [3H]nisoxetine to NETs. The down-regulation of NETs was paralleled by a DMI-induced reduction in the uptake of [3H]norepinephrine in intact PC12 cells. It can be inferred from these data that inhibitors of the NET can down-regulate NETs directly, and do so in the absence of changes in the synaptic concentration of norepinephrine.

When grown for seven days in a medium containing nerve growth factor (100 ng/ml), 10% horse serum and 5% fetal bovine serum PC12 cells stopped dividing, extended neurites and assumed a neuronal phenotype. Withdrawal of nerve growth factor from these cells resulted in loss of neurites and apoptotic changes in many cells. The apoptotic changes were exacerbated if the cells were also exposed to 1-2 microM S-100, a calcium binding protein purified from bovine brain. After exposure to S-100, the PC12 cells underwent characteristic apoptotic changes. Within 2 in neurites retracted, the cell body shrunk and submembranous accumulation of condensed cytoplasmic material was observed. DNA ladders were present after 24-48 h and 60% of the cells became hypodiploid after 72 h. S-100 induced apoptosis by binding to specific sites (Kd = 189 nM) on PC12 cells and this caused a rise in [Ca2+]i due to a transmembrane capacitative flux followed by the depletion of internal stores. This increase was reversed if 5 microM nifedipine, a specific L-type Ca2+ channel inhibitor, was added to the medium after S-100 and completely abolished if the cells were pretreated with 5 microM thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. The presence of nerve growth factor in the culture medium completely blocked the apoptotic changes induced by S-100, probably due to interaction of nerve growth factor and S-100 at the same binding sites. These data indicate that nerve growth factor not only prevents apoptosis during cell development, but also apoptosis induced by endogenous substances such as S-100.

3,4-Dihydroxyphenylglycolaldehyde (DOPEGAL) is the monoamine oxidase A (MAO-A) metabolite of norepinephrine (NE) and epinephrine (Epi). Oxidative metabolites of amines are predicted toxins. In this study we determine the toxicity of DOPEGAL, its tautomer 2',3,4-trihydroxyacetophenone (THAP) as well as NE, Epi and their oxidative and methylated metabolites in cultures of differentiated PC-12 cells. At 59.5 microM DOPEGAL, THAP and Epi, but not NE or other NE or Epi metabolites decreased PC-12 cells by 43.8%, 26.7% and 16.8% respectively. DOPEGAL toxicity was concentration and time dependent. Possible implications for degenerative diseases are discussed.

The molecular mechanism of cell death due to hypoxia has not been elucidated. Our recent observations that overexpression of the anti-apoptotic proto-oncogene bcl-2 and a bcl-2-related gene, bcl-x, prevents hypoxic cell death suggest that hypoxia induces apoptosis. Using electron microscopy and confocal and nonconfocal fluorescence microscopy, we show here that hypoxia does, in fact, induce both necrosis and apoptosis, and that the proportion of these two modes is highly dependent on the cell type. Overexpression of Bcl-2 or Bcl-Xl blocks hypoxia-induced apoptosis in a dose-dependent manner.

Previously we reported the results of a series of experimental tests using PC-12 cells to examine the biological effect of prescribed combinations of both nerve growth factor and magnetic fields. Because our assay of the PC-12 cells is based on binary classification of the cells following treatment, our data might be expected to have a binomial distribution. However, our data consistently show a smaller variability than that predicted by the binomial distribution model. In this paper, we examine some possible reasons for this reduction in variability in our results.

Blackman et al. [1994] describe an experiment that purports to show that weak 45 Hz magnetic fields inhibit the growth of neurites from PC-12 cells treated with a growth stimulation factor. I present a statistical analysis of the data in that paper that shows that the data were corrupted in some way; hence, the results are invalid.

This investigation examined if lithium, the primary therapeutic treatment for bipolar affective disorder, modulated the levels of selected signal transduction proteins in PC12 cells. Nerve growth factor (NGF) induced differentiation of PC12 cells, and after 12 days of NGF treatment there were large increases in the levels of the heterotrimeric G protein subunits alpha o1, alpha i1, beta, and alpha s, small increases in those of alpha i2 and alpha q, and a slight decrease in that of alpha o2. Lithium (1 mM, equivalent to the therapeutic concentration) selectively reduced NGF-induced increases in levels of G protein subunits, generally having the greatest inhibition on those that were increased the most by NGF. Lithium at 5 mM had greater inhibitory effects than 1 mM lithium on NGF-induced increases in levels of G proteins, but neither concentration of lithium affected the induction of the cytoskeletal protein beta-tubulin. Examination of other proteins involved in signal transduction revealed that 12 days of NGF treatment increased the level of protein kinase C-alpha, but not those of the beta, epsilon, or zeta subtypes, and did not alter the levels of beta, gamma, or delta phospholipase C. Pretreatment with lithium inhibited the increase in content of protein kinase C-alpha induced by NGF but had little effect on the proteins not responsive to NGF except for decreasing the levels of protein kinase C-epsilon. The inhibitory effect of lithium was found not to be due to inhibition of NGF-induced tyrosine phosphorylation, which was unaffected by 5 mM lithium, or to inositol depletion. In summary, use of the dynamic system of NGF-induced PC12 cell differentiation provided a sensitive model in which to identify signal transduction proteins that were influenced by lithium treatment. The large changes caused by a therapeutically equivalent concentration of lithium lend support to the proposal that the selective inhibitory effects of lithium on subtypes of G proteins and protein kinase C may be important therapeutic targets.