Morphological differentiation of rat pheochromocytoma cells (PC12 cells) by electric stimulation.

The effects of electric stimulation on the morphological differentiation of PC12 cells are described. PC12 cells were stimulated with the 'theta' (4-7 Hz electroencephalogram (EEG) rhythm) pattern-electric stimulation, which was known to elicit stable long-term potentiation (LTP) in the CA1 region of the hippocampus. The stimulation induced the neurite outgrowth of PC12 cells, as well as nerve growth factor (NGF). This result suggests that the electric signal has a differentiating potential equivalent to the receptor-ligand interaction.     

Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation

Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations. Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death. To accomplish this, we have utilized rat pheochromocytoma (PC12) cells as a model since they possess much of the biochemical machinery associated with dopaminergic neurons. Mn, like nerve growth factor (NGF), can induce neuronal differentiation of PC12 cells but Mn-induced cell differentiation is dependent on its interaction with the cell surface integrin receptors and basement membrane proteins, vitronectin or fibronectin. Similar to NGF, Mn-induced neurite outgrowth is dependent on the phosphorylation and activation of the MAP kinases, ERK1 and 2 (p44/42). Unlike NGF, Mn is also cytotoxic having an IC50 value of approximately 600 microM. Although many apoptotic signals are turned on by Mn, cell death is caused ultimately by disruption of mitochondrial function leading to loss of ATP. RT-PCR and immunoblotting studies suggest that some uptake of Mn into PC12 cells depends on the divalent metal transporter 1 (DMT1). DMT1 exists in two isoforms resulting from alternate splicing of a single gene product with one of the two mRNA species containing an iron response element (IRE) motif downstream from the stop codon. The presence of the IRE provides a binding site for the iron response proteins (IRP1 and 2); binding of either of these proteins could stabilize DMT1 mRNA and would increase expression of the +IRE form of the transporter. Iron and Mn compete for transport into PC12 cells via DMT1, so removal of iron from the culture media enhances Mn toxicity. The two isoforms of DMT1 (+/-IRE) are distributed in different subcellular compartments with the -IRE species selectively present in the nucleus of neuronal and neuronal-like cells.     

Chronic exposure to inorganic lead increases high-threshold voltage-gated calcium currents in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma (PC12) cells were exposed to lead acetate (0, 10, 25 and 50 μM) in their growth media for up to 12 weeks. High-threshold voltage-gated calcium currents were recorded each week from nerve growth factor-differentiated PC12 cells using the whole-cell patch-clamp technique. Chronic exposure for 1 month did not modify peak or sustained calcium current amplitudes in lead-treated cells when compared to sister control cultures. Two month exposure to 25 and 50 μM significantly increased peak and sustained calcium current amplitudes, while 10 μM had little effect. During the third month of exposure, peak and sustained calcium current amplitudes remained increased in the cells exposed to 25 and 50 μM lead acetate. By the end of the second month of exposure to 25 and 50 μM lead acetate, the voltage at which maximal current amplitude was attained shifted from +10 mV to 0 mV. The observed effects of toxicologically relevant lead concentrations on high-threshold calcium currents in chronically exposed mammalian cells provide further support for the notion that at least one cellular target of the heavy metal's neurotoxic action may be the voltage-gated calcium channel.     

Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells

Mn²⁺ has been shown to promote cell–substrate adhesion and cell spreading in many cell culture systems. In this study, we present data demonstrating that Mn²⁺ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells. In the presence of 1.0 mM MnCl₂, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes. These morphological effects of Mn²⁺ were both time- and dose-dependent. In the presence of cycloheximide, a protein synthesis inhibitor, both Mn²⁺-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn²⁺ to take place. Of the other divalent cations tested, Mg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, and Zn²⁺ were ineffective, and only Co²⁺ partially mimicked the effects of Mn²⁺. Although Mn²⁺-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth. The neurite outgrowth-promoting effects of Mn²⁺ were distinct from those of nerve growth factor in that the response to Mn²⁺ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites. Mn²⁺ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells. In cells grown in serum-free defined medium, Mn²⁺ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata. Similarly, Mn²⁺ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only. The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn²⁺ on PC12 cells. These findings are consistent with a hypothesis that Mn²⁺-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects. © 1993 Wiley-Liss, Inc.     

A convenient bioassay for NGF using a new subline of PC12 pheochromocytoma cells (PC12D)

A useful bioassay for nerve growth factor (NGF) has been developed, based on the rapid outgrowth of neurites (within 24 h) from cells of a new subclone of PC12 cells (PC12D) in response to NGF. The sensitivity is similar to that of other bioassay systems that the the sensory ganglia of chick embryos of primed PC12 cells.The assay is readily adaptable for the purification of NGF and for the determination of levels of NGF in tissue, as shown by a comparison of results from this assay to the data obtained by an immunological assay.     

Cocaine inhibits NGF-induced PC12 cells differentiation through D(1)-type dopamine receptors

In utero cocaine exposure can adversely affect CNS development. Previous studies showed that cocaine inhibits neuronal differentiation in a dose-dependent fashion in nerve growth factor (NGF)-stimulated PC12 cells. Cocaine binds with high affinity to several neurotransmitter transporters, resulting in elevated neurotransmitter levels in nerve endings. To determine if cocaine inhibits neurite outgrowth through the effects of these neurotransmitters, we applied dopamine, norepinephrine, serotonin, and acetylcholine to NGF-induced PC12 cells. Dopamine was the only neurotransmitter to inhibit neurite outgrowth significantly in a dose-dependent pattern without affecting cell viability. Norepinephrine and acetylcholine did not affect neurite outgrowth, while serotonin enhanced it. Furthermore, GBR 12909, a potent dopamine transporter (DAT) inhibitor, yielded similar effects. We then showed PC12 cells express D(1) and D(2) receptors and DAT proteins. Dopamine uptake measured over time was significantly blocked by cocaine and GBR 12909 which may result in elevated extracellular dopamine. The role of dopamine receptors in PC12 differentiation was further examined by using D(1) and D(2) specific receptor agonists. Only the D(1) agonist, SKF-38393, had a significant dose-dependent inhibitory effect. In addition, a D(1) antagonist produced significant recovery of neurite outgrowth in cocaine-treated cells. These findings suggest that cocaine inhibitory effects on neuronal differentiation are mediated through its binding to the dopamine transporter, resulting in increased dopamine level in the synapses. Subsequently, up regulation of D(1) receptors alters NGF signaling pathways.     

The synthetic cannabinoid HU-210 attenuates neural damage in diabetic mice and hyperglycemic pheochromocytoma PC12 cells

Diabetic neuropathy (DN) is a common complication of diabetes mellitus resulting in cognitive dysfunction and synaptic plasticity impairment. Hyperglycemia plays a critical role in the development and progression of DN, through a number of mechanisms including increased oxidative stress. Cannabinoids are a diverse family of compounds which can act as antioxidative agents and exhibit neuroprotective properties. We investigated the effect of the synthetic cannabinoid HU-210 on brain function of streptozotocin (STZ)-induced diabetic mice. These animals exhibit hyperglycemia, increased cerebral oxidative stress and impaired brain function. HU-210, through a receptor independent pathway, alleviates the oxidative damage and cognitive impairment without affecting glycemic control. To study the neuroprotective mechanism(s) involved, we cultured PC12 cells under hyperglycemic conditions. Hyperglycemia enhanced oxidative stress and cellular injuries were all counteracted by HU-210-in a dose dependent manner. These results suggest cannabinoids might have a therapeutic role in the management of the neurological complications of diabetes.     

Differentiation of PC12 pheochromocytoma cells by sodium butyrate

PC12 pheochromocytoma cells cease dividing and show increased cell-cell and cell-substratum adhesion in response to treatment with sodium butyrate. These changes are accompanied by the rapid appearance of neuron-specific enolase, an APUD cell marker. However, neurofilament proteins, markers for neuronal differentiation, are not induced. These results suggest that sodium butyrate induces differentiation in the PC12 cell line, perhaps along the chromaffin cell pathway.     

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.     

Acetyl-L-Carnitine arginyl amide (ST857) increases calcium channel density in rat pheochromocytoma (PC12) cells

We used the patch clamp technique to study the effect of acetyl-L-carnitine arginyl amide (ALCAA) and of nerve growth factor (NGF) on availability of L-type Ca²⁺ channels in rat pheochromocytoma (PC12) cells maintained in defined medium. Channel availability was measured as number of channels in the patch × the probability of opening (n^.P₀). In patches from control cells, cells exposed to NGF (10 ng/ml) for six days, and cells exposed to ALCAA (1 mM) for six days, n^.P₀, measured during 200–240 ms pulses to -10 mV (holding potential, ?60 mV), was 0.102 ± 0.089 (5 cells), 0.173 ± 0.083 (5 cells), and 0.443 ± 0.261 (7 cells), respectively. The 4.3-fold increase for the ALCAA-treated cells was significantly different from control (P < 0.05), whereas that for the NGF-treated cells was not. For the same conditions, the maximum number of superimposed openings at ?10 mV was 1.3 ± 0.5 (6 cells), 1.6 ± 0.5 (8 cells), and 3.3 ± 1.8 (8 cells), with the value for the ALCAA-treated cells being significantly different from control (P < 0.001). Additional analysis showed that the distribution of channel open times, the time constants, and the voltage dependence of activation were not changed by prolonged exposure to ALCAA. Short-term exposure to both ALCAA as well as to the parent compound, acetyl-L-carnitine (ALCAR), did not cause an increase but rather a decrease in n^.P₀, and this short-term effect of both compounds was blocked by neomycin, an inhibitor of phospholipase C. Together, our findings are consistent with the interpretation that short-term exposure to ALCAA inhibits Ca²⁺ channel activity, possibly by increasing intracellular Ca²⁺, and that long-term exposure causes an increase in Ca²⁺ channel density, possibly by increasing channel expression, with no change in Ca²⁺ channel properties. © 1995 Wiley-Liss, Inc.     

The I(1)-imidazoline receptor in PC12 pheochromocytoma cells reverses NGF-induced ERK activation and induces MKP-2 phosphatase

We sought to further elucidate signal transduction pathways for the I(1)-imidazoline receptor in PC12 cells and their interaction with the well-characterized signaling events triggered by nerve growth factor (NGF) in these cells. Stimulation of the I(1)-imidazoline receptor with moxonidine, a centrally acting antihypertensive, increased by greater than two-fold the proportion of ERK-1 and ERK-2 in the phosphorylated active form. Similarly, NGF elicited a five-fold increase in activated ERKs. Surprisingly, treatment of NGF-treated cells with moxonidine completely reversed activation of ERK. Moxonidine-induced inhibition of ERK activation in NGF-treated cells was dose-dependent, followed a limited time course and could be blocked by the I(1)-antagonist efaroxan. These data suggested possible deactivation of ERK by specific phosphatases. Therefore, we assayed levels of MKP-2, a dual specificity phosphatase whose substrates include ERK. Moxonidine and NGF both increased levels of MKP-2 by three-fold. These effects were additive, as both agents together increased MKP-2 by a total of six-fold. Moxonidine-induced induction of MKP-2 was time- and dose-dependent and could be blocked by the I(1)-antagonist efaroxan or by D609, an inhibitor of phosphatidylcholine-selective phospholipase C known to block downstream signaling events coupled to I(1)-receptors. Thus, I(1)-receptors can abrogate the primary signaling cascade activated by NGF, most likely by increasing levels of a specific phosphatase to return dually phosphorylated ERK to its unphosphorylated state.     

Activation of ERK1 and ERK2 is required for manganese-induced neurite outgrowth in rat pheochromocytoma (PC12) cells.

Mn(2+) treatment has been shown to promote neurite outgrowth in rat pheochromocytoma (PC12) cells in a time- and dose-dependent manner. This process is mediated through the interactions of extracellular matrix (ECM) proteins and integrin receptors. Studies were performed to determine whether the phosphorylation of the MAP kinases, ERK1 and 2, is required for Mn(2+)-induced neurite outgrowth. A time- and dose-dependent increase in phosphorylation of both ERK1 and 2 was observed upon treatment of PC12 cells with Mn(2+). Phosphorylation of the ERKs occurred as early as 2 hr after initiating treatment, with a maximum increase occurring at approximately 24 hr. Inhibition of MEK with the specific inhibitor, PD98059, blocked the phosphorylation of ERK1 and 2 and increased Mn(2+) toxicity. When cells were grown in serum-free defined medium, Mn(2+)-induced phosphorylation of ERK1 and ERK2 occurred in cells grown on surfaces treated with growth serum or fibronectin but not on surfaces treated with poly-L-lysine. In addition, the pentapeptide GRGDS, which blocks RGD-mediated interactions, inhibited Mn(2+)-induced phosphorylation of ERK1 and 2. The Mn(2+)-induced increase in phosphorylated ERK1 and 2 was not seen in a PC12 cell line that does not respond to Mn(2+). These data support the hypothesis that integrin-mediated activation of the MAPK signal transduction pathway leading to the activation of ERK1 and 2 is required for Mn(2+)-induced PC12 differentiation and neurite outgrowth.     

Purine-induced alterations of dopamine metabolism in rat pheochromocytoma PC12 cells

Studies with cerebrospinal fluid from subjects with Parkinson's disease suggest that purine abnormalities may be present in this disorder. The effects of purines on dopamine metabolism have not been characterized, though adenosine is known to inhibit dopaminergic neurotransmission. In this study, dopamine, its precursor 3,4-dihydroxyphenylalanine (DOPA), and its degradation products 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured in rat pheochromocytoma PC12 cells following 24-h incubation with 5, 50, and 500 microM adenosine, adenine, guanosine, guanine, hypoxanthine, xanthine, and uric acid. Incubation with adenosine increased DOPA, DOPAC, and HVA, while adenine treatment decreased DOPA. Guanosine (500 microM) decreased DOPA, dopamine, and DOPAC, while lower concentrations increased DOPAC and HVA. Incubation with guanine decreased dopamine, and xanthine decreased dopamine and DOPAC. Hypoxanthine and uric acid exerted minimal effects. These results indicate that purines exert a variety of effects on dopamine metabolism. The influence of purine metabolism on the dopaminergic deficit in the Parkinsonian brain merits further investigation.     

Survival and differentiation within the adult mouse striatum of grafted rat pheochromocytoma cells (PC12) genetically modified to express recombinant beta-NGF.

Rat pheochromocytoma PC12 cells were genetically modified in vitro to express recombinant beta-nerve growth factor (beta-NGF) using a replication-deficient retroviral vector carrying the mouse beta-NGF gene and subsequently implanted into the striatum of a mouse model of Parkinson's disease. The fate of the genetically modified PC12 cells (PC12N.8) was assessed at varying times postimplantation by studying immunoreactivity (IR) to tyrosine hydroxylase (TH) or the rat NGF receptor (NGFR). In vitro, the genetically modified PC12 cells displayed a neuronal morphology in the absence of exogenous NGF which was characterized by extensive neurite outgrowth. In addition, the genetically modified PC12 displayed a catecholaminergic phenotype in vitro as assessed by TH-IR. Following implantation into the striatum, the survival of PC12N.8 cells was limited. Surviving cells could be identified by NGFR-IR, but not by TH-IR. In addition, PC12N.8 cells with a neuronal morphology similar to that observed in vitro were only rarely observed in vivo. No tumors were observed in PC12N.8 graft recipients up to 30 days postimplantation. In contrast, intrastriatal tumors were observed in 50% of the PC12 cell recipients. These data demonstrate that PC12 cells genetically modified in vitro to synthesize beta-NGF do not revert to the mitotic phenotype of the parent PC12 cell line following implantation into the adult striatum, an observation that suggests that these cells may continue to express recombinant beta-NGF in vivo. The data further suggest that the genetically modified PC12 cells lose the catecholaminergic phenotype following implantation into the striatal parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of bradykinin- and ATP-activated Ca(2+)-permeable channels in rat pheochromocytoma (PC12) cells.

Membrane currents activated by bradykinin (500 nM) and by extracellular ATP (50 microM) were studied in voltage-clamped, NGF-treated rat pheochromocytoma (PC12) cells. Under quasiphysiological ionic conditions, both substances caused an outward current due to opening of Ca(2+)-activated K+ channels. Bradykinin caused an additional inward current that could be studied after blockade by internal Cs+ of the initial transient outward current. The inward current became larger when the extracellular Ca2+ concentration was increased. Neither inositol-1,4,5-trisphosphate, dioctanoylglycerol, phorbol 12-myristat 13-acetate, forskolin, GTP, GTP-gamma-S, or pretreatment with pertussis toxin affected this current component. Increasing the internal Ca buffer concentration [EGTA or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid] from 1 to 10 mM had no effect on the inward current as long as the free [Ca2+]i was kept constant. However, it was modulated by the resting free [Ca2+]i. Elevation of [Ca2+]i from nominally 0 to 60 or to 180 nM increased the bradykinin-induced average peak current density from 0.14 to 1.04 or to 2.29 pA/pF, respectively. This regulation may depend on a calmodulin-dependent pathway, since CGS 9343B, a calmodulin inhibitor, blocked the effect of elevated [Ca2+]i. With ATP as an agonist, outward current was preceded by a large inward current that was partially blocked by extracellular Ca2+ in the millimolar range. Extracellular Ca2+ was also found to reduce the single-channel conductance estimated from outside-out patches treated with ATP.     

The effect of exogenous human ras and myc oncogenes in morphological differentiation of the rat pheochromocytoma PC12 cells

An electroporation technique was employed to study the effect of oncogenes H-ras and c-myc after their short-term expression in the rat pheochromocytoma PC12 cells. It was found that within 6 days after electroporation the mutant T24 H-ras 1 gene induced differentiation of PC12 cells whereas the c-myc blocked NGF-induced differentiation. The induction of differentiation by the T24 H-ras gene may suggest a physiological role of the ras gene in cell differentiation as well as in cell proliferation.