Pheochromocytoma 12 Research Articles

Nitric oxide serves as an endogenous regulator of neuronal adenosine A1 receptor expression

Nitric oxide (NO) radicals are produced during normal cellular function, after tissue injury, and in response to immune system activation during infection. The transformation of NO to peroxynitrite is essential for mediating some of its physiological and/or cytotoxic actions. As the expression of the adenosine A1 receptor (A1AR) is regulated by oxidative stress, we evaluated the role of NO in the regulation of A1AR expression, a G protein-coupled receptor involved in cytoprotection in the central nervous system. Administration of the NO donor, S-nitrosylpenicillamine (SNAP), to pheochromocytoma 12 (PC12) cells increased A1AR protein in a time- and dose-dependent manner, with maximal induction observed with 20 microm SNAP at 24 h. The response to SNAP was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (C-PTIO), and by the inhibition of nuclear factor-kappaB (NF-kappaB), implicating this transcription factor in the regulatory process. In addition SNAP also increased the degradation of Inhibitory kappaB-alpha (IkappaB-alpha), a marker of NF-kappaB activation. Furthermore, the induction of inducible nitric oxide synthase (iNOS) by lipopolysaccharide increased A1AR in PC12 cells and in mice, whereas the inhibition of NOS activity suppressed this response. We conclude that NO, via the activation of NF-kappaB, serves as an endogenous regulator of A1AR, and speculate that the induction of the A1AR could counteract the cytotoxicity of NO.

Moving PIP 3 on Microtubules

Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), the product of phosphatidylinositol 3-kinase (PI3K), contributes to the establishment of cell polarity. Horiguchi et al. provide evidence that PIP 3 not only is produced by local activation of PI3K at the plasma membrane but also may be produced at internal membranes and transported by PIP 3 -containing vesicles on microtubules to the growing tips of neuronal projections. First, they determined that GAKIN (guanylate kinase-associated kinesin, also known as KIF13B) interacted with PIP 3 binding protein (PIP 3 BP, also known as centaurin-α) through the GAKIN FHA domain. In vitro GAKIN and PIP 3 BP mediated the movement of PIP 3 liposomes on microtubules. In PC12 (pheochromocytoma 12) cells and culture hippocampal neurons, tagged GAKIN, tagged PIP 3 BP, and a marker for PIP3 were colocalized at the tips of neurites; in hippocampal cells, these three molecules were most abundant in the longest neurite, the axon. Overexpression of a dominant-negative form of GAKIN (with the motor domain deleted) in PC12 cells decreased the abundance of PIP 3 at neurite tips. In hippocampal neurons, overexpression of wild-type GAKIN or dominant-negative GAKIN disrupted the formation of morphologically distinct axon-dendrite structure and produced cells with multiple, highly branched neurites. The authors suggest that PIP 3 produced at internal membranes, for example at internalized receptors, or PIP 3 produced at the cell body may contribute to cell polarity through transport along microtubules to the growth cone. K. Horiguchi, T. Hanada, Y. Fukui, A. H. Chishti, Transport of PIP 3 by GAKIN, a kinesin-3 family protein, regulates neuronal cell polarity. J. Cell Biol. 174 , 425-436 (2006). [Abstract] [Full Text]

Inhibition of Apoptosis by P2Y2Receptor Activation: Novel Pathways for Neuronal Survival

Cell survival is an essential function in the development and maintenance of the nervous system. We demonstrate here a previously unappreciated role for extracellular nucleotide signaling through the P2Y2 receptor in the survival of neurons: PC12 (pheochromocytoma 12) cells and dorsal root ganglion neurons are protected from serum starvation-induced apoptosis by ATP, UTP, and ATPgammaS, an effect mediated via P2Y2 receptors, as demonstrated by small interfering RNA and genetic knock-out models. This protection occurs independently of neurophin signaling but requires Src activation of ERK (extracellular signal-regulated kinase) and Akt. Moreover, ATPgammaS and NGF act synergistically to enhance neuronal survival through enhanced TrkA signaling. The results, which define a novel mechanism for inhibition of apoptosis, implicate parallel, interacting systems--extracellular nucleotides/P2Y2 receptors and neurotrophin/TrkA--to sustain neuronal survival.

The transcription factor Yin Yang 1 is an activator of BACE1 expression

The beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a prerequisite for the generation of beta-amyloid peptides, the principle constituents of senile plaques in the brains of patients with Alzheimer's disease (AD). BACE1 expression and enzymatic activity are increased in the AD brain, but the regulatory mechanisms of BACE1 expression are largely unknown. Here we show that Yin Yang 1 (YY1), a highly conserved and multifunctional transcription factor, binds to its putative recognition sequence within the BACE1 promoter and stimulates BACE1 promoter activity in rat pheochromocytoma 12 (PC12) cells, rat primary neurones and astrocytes. In rat brain YY1 and BACE1 are widely expressed by neurons, but there was only a minor proportion of neurones that co-expressed YY1 and BACE1, suggesting that YY1 is not required for constitutive neuronal BACE1 expression. Resting astrocytes in the untreated rat brain did not display either YY1 or BACE1 immunoreactivity. When chronically activated, however, astrocytes expressed both YY1 and BACE1 proteins, indicating that YY1 is important for the stimulated BACE1 expression by reactive astrocytes. This is further emphasized by the expression of YY1 and BACE1 by reactive astrocytes in proximity to beta-amyloid plaques in the AD brain. Our observations suggest that interfering with expression, translocation or binding of YY1 to its BACE1 promoter-specific sequence may have therapeutic potential for treating patients with AD.

Orally Active Neurotrophin-Enhancing Agent Protects Against Dysfunctions of the Peripheral Nerves in Hyperglycemic Animals

Biological substances with neurotrophic activities, such as nerve growth factor (NGF) and monosialoganglioside GM1, have been considered as agents for diabetic peripheral neuropathy. Because recent studies have suggested that decreased availability of these substances might contribute to the pathogenesis of diabetic peripheral neuropathy, some clinical trials of NGF for diabetic peripheral neuropathy have been conducted and have led to mixed conclusions. The major reasons were its limited delivery to the nervous system and adverse effects induced by subcutaneous injection, which was necessary because NGF is a polypeptide. The current study investigates whether an orally active sialic acid derivative, MCC-257, has neuroprotective properties in diabetic peripheral nerves. MCC-257 augmented NGF activity in cultured dorsal root ganglia and PC12 (pheochromocytoma 12) cells. Treatment with MCC-257 elevated NGF levels in the sciatic nerve, accompanied by improvement in nerve conduction velocity in streptozotocin-induced diabetic animals. More importantly, MCC-257 ameliorated small fiber dysfunctions, including thermal hypoalgesia, substance P content, and histopathological innervation in the plantar skin of diabetic animals. Thus, the orally active neurotrophin enhancer provides a new option for the clinical treatment of diabetic peripheral neuropathy.

The MAP kinase pathway is upstream of the activation of GSK3β that enables it to phosphorylate MAP1B and contributes to the stimulation of axon growth

In pheochromocytoma 12 (PC12) cells and sympathetic neurons, nerve growth factor (NGF) engagement with the tropomyosin-related tyrosine kinase (TrkA) receptor activates the serine/threonine kinase glycogen synthase kinase 3β (GSK3β), enabling it to phosphorylate the microtubule-associated protein 1B (MAP1B). GSK3β phosphorylation of MAP1B acts as a molecular switch to regulate microtubule dynamics in growing axons, and hence the rate of axon growth. An important question relates to the identification of the upstream pathway linking the activation of GSK3β with TrkA engagement. TrkA can utilise a number of intracellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3 kinase (PI3K) pathway. We now show, using pharmacological inhibitor studies of PC12 cells and sympathetic neurons in culture and in vitro kinase and activation assays, that the MAPK pathway, and not the PI3K pathway, links NGF engagement with the TrkA receptor to GSK3β activation in PC12 cells and sympathetic neurons. We also show that activated GSK3β is a small fraction of the total GSK3β present in developing brain and that it is not part of a multiprotein complex. Thus, NGF drives increased neurite growth rates partly by coupling the MAPK pathway to the activation of GSK3β and thereby phosphorylation of MAP1B.

Direct Measurement of Multiple mRNAs in Nerve Growth Factor-Induced PC12 Cells Using Electrophoretic Tags to Monitor Biomarkers of Neuronal Differentiation in 96-Well Format

Pheochromocytoma-12 (PC12) cells recapitulate the program of neuronal differentiation by developing neurites after about 12 days of nerve growth factor (NGF) treatment. This model can be used to evaluate the neuroprotective/neurotrophic effect of compounds. Specific mRNAs such as cfos and c-jun are early biomarkers of the irreversible commitment into the differentiation program as they appear after only 30-40 min of NGF treatment. Monitoring the level of these mRNAs instead of the neurite outgrowth dramatically reduces the time needed to identify the drug potential of compounds. The electrophoretic tags, or eTag reporters (ACLARA Biosciences, Inc., Mountain View, CA), are a new class of fluorescent reporters that have unique migration properties in capillary electrophoresis, which allows for their separation and identification. (The eTag Multiplex Invader Assay and products incorporate Invader technology and Cleavase enzyme licensed for use from Third Wave Technologies, Inc. [Madison, WI] for multiplexed gene expression applications.) Each eTag molecule used begins as a phosphoramidite that is incorporated into a specific oligonucleotide using standard oligonucleotide synthesis procedures. A set of distinct probes labeled with different eTag molecules can then be mixed together to simultaneously quantify the levels of different mRNAs from the same sample. When compared to existing methods for measuring multiplexed gene expression from the same sample, the eTag assay allows a direct quantification of the mRNA from cells without any extraction/purification and still provides multiplexing capability, high sensitivity, miniaturization, and reproducibility compatible with medium-throughput screening methods. The eTag technology was used to simultaneously measure the level of expression of four mRNAs-c-fos, c-jun, c-myc, and gapdh-in NGF-treated PC12 cells in a standard 96-well format. The experimental data shown here demonstrate the use of eTag technology as a new screening tool, which uniquely combines robustness, sensitivity, multiplexing capability, and direct measurement of mRNA without any sample preparation steps, such as RNA extraction/purification or a reverse transcription step.

The Role of Palmitoylation in Functional Expression of Nicotinic α7 Receptors

Neuronal alpha-bungarotoxin receptors (BgtRs) are nicotinic receptors that require as yet unidentified post-translational modifications to achieve functional expression. In this study, we examined the role of protein palmitoylation in BgtR expression. BgtR alpha7 subunits are highly palmitoylated in neurons from brain and other cells capable of BgtR expression, such as pheochromocytoma 12 (PC12) cells. In PC12 cells, alpha7 subunits are palmitoylated with a stoichiometry of approximately one palmitate per subunit, and inhibition of palmitoylation blocks BgtR expression. In cells incapable of BgtR expression, such as human embryonic kidney cells, alpha7 subunits are not significantly palmitoylated. However, in these same cells, chimeric subunits with the N-terminal half of alpha7 fused to the C-terminal half of serotonin-3A receptor (alpha7/5-HT3A) subunits form functional BgtRs that are palmitoylated to an extent similar to that of BgtRalpha7 subunits in PC12 cells. Palmitoylation of PC12 and alpha7/5-HT3A BgtRs occurred during assembly in the endoplasmic reticulum (ER). In conclusion, our data indicate a function for protein palmitoylation in which palmitoylation of assembling alpha7 subunits in the ER has a role in the formation of functional BgtRs.

L-Glutamate in the extracellular space regulates endogenous d-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that d-aspartate ( d-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of d-Asp homeostasis in these cells. In this communication, we show that l-glutamate (Glu) and l-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous d-Asp in MPT1 cells. d-Asp is apparently in dynamic homeostasis, whereby endogenous d-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an l-Glu transporter. Under these conditions, l-Glu and its transporter substrates in the medium may competitively inhibit the uptake of d-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that dl-TBOA, a well-established l-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.

Heuristics-Based integrated decisions for logistics network systems

The graphene oxide (GO) has attracted tremendous attention in biomedical fields. In order to combine the unique physicochemical properties of GO nanosheets with topological structure of aligned nanofibrous scaffolds for nerve regeneration, the GO nanosheets were coated onto aligned and aminolyzed poly-l-lactide (PLLA) nanofibrous scaffolds. Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) revealed that the surface of aligned PLLA nanofibers after being coated with GO became rougher than those of the aligned PLLA and aminolyzed PLLA nanofibrous scaffolds. The GO nanosheets did not destroy the alignment of nanofibers. The characterizations of X-ray photoelectron spectroscopy (XPS) and water contact angle displayed that the aligned PLLA nanofibrous scaffolds were introduced with hydrophilic groups such as NH2, COOH, and OH after aminolysis and GO nanosheets coating, showing better hydrophilicity. The GO-coated and aligned PLLA nanofibrous scaffolds significantly promoted Schwann cells (SCs) proliferation with directed cytoskeleton along the nanofibers compared with the aligned PLLA and aminolyzed PLLA nanofibrous scaffolds. These scaffolds also greatly improved the proliferation of rat pheochromocytoma 12 (PC12) cells, and significantly promoted their differentiation and neurite growth along the nanofibrous alignment in the presence of nerve growth factor (NGF). This type of scaffolds with nanofibrous surface topography and GO nanosheets is expected to show better performance in nerve regeneration.Recovery of damaged nerve functions remains a principal clinical challenge in spite of surgical intervention and entubulation. The use of aligned fibrous scaffolds provides suitable microenvironment for nerve cell attachment, proliferation and migration, enhancing the regeneration outcome of nerve tissue. Surface modification is generally required for the synthetic polymeric fibers by laminin, fibronectin and YIGSR peptides to stimulate specific cell functions and neurite outgrowth. Yet these proteins or peptides present the poor processibility, limited availability, and high cost, influencing their application in clinic. In this work, we combined GO nanosheets and topological structure of aligned nanofibrous scaffolds to direct cell migration, proliferation, and differentiation, and to induce neurite outgrowth for nerve regeneration. The GO coating improved several biomedical properties of the aligned PLLA nanofibrous scaffolds including surface roughness, hydrophilicity and promotion of cells/material interactions, which significantly promoted SCs growth and regulated cell orientation, and induced PC12 cells differentiation and neurite growth. The design of this type of structure is of both scientific and technical importance, and possesses broad interest in the fields of biomaterials, tissue engineering and regenerative medicine.

Group IIA secretory phospholipase A 2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons

Recent evidence shows that secretory phospholipase A 2 (sPLA 2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA 2 on vesicle exocytosis. External application of group IIA sPLA 2 (purified crotoxin subunit B or purified human synovial sPLA 2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA 2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA 2 was dependent on calcium and sPLA 2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA 2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA 2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA 2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA 2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA 2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

499 CT-based three-dimensional intracavitary brachytherapy planning in cervix cancer: Is it always better than conventional planning?

Our previous research revealed that rifampicin could protect PC12 (pheochromocytoma 12) cells from rotenone-induced cytotoxicity by reversing the aggregation of α-synuclein. Furthermore, increasing evidence indicated that the misfolded α-synuclein with SUMOylation, an important protein posttranslational modification, was easier to solubilize and was less toxic. Here, we investigated whether rifampicin could stabilize α-synuclein and prevent rotenone-induced PC12 cells from undergoing apoptosis by enhancing SUMOylation of α-synuclein. The expression of SUMO1 and SUMO2/3, the two main proteins responsible for the SUMOylation modification in PC12 cells, were detected by western blotting. Co-immunoprecipitation was performed to compare qualitatively the SUMOylation modification of α-synuclein. The cell viability and apoptosis rate were measured by a CCK-8 assay kit and flow cytometry, respectively. We targeted Ubc9 as a key enzyme in the SUMOylation modification pathway and knocked down the UBC9 gene using a short interfering RNA. Treatment with 150 μmol/L rifampicin, increased the expressions of SUMO1 and SUMO2/3 in cells by 1.5 times compared with the control group; meanwhile, the cell viability of rotenone-induced cells increased from 20 to 80% (P < 0.05). In addition, the increased SUMOylation activity in the cells stimulated by rifampicin was observed 18 h earlier compared with cells treated by rotenone alone. SUMOylation of α-synuclein was more significant in rifampicin-treated cells and Ubc9 upregulated cells. However, the same phenomenon and the protective effect of rifampicin were reversed after UBC9 knockout. In conclusion, rifampicin might reduce the cytotoxicity of rotenone-induced PC12 cells by promoting SUMOylation of α-synuclein.

Synaptotagmin Regulation of Secretory Mode

Vesicular secretion of neurotransmitter can take place through two distinct mechanisms: full fusion, in which a fusion pore opens and dilates and the vesicle completely releases its contents; and kiss-and-run, in which the fusion pore opens but does not dilate. Wang et al. performed amperometric measurements of catecholamine release in cultured pheochromocytoma 12 (PC12) cells to distinguish between these two forms of secretion and to investigate the role of synaptotagmins (Syts) in regulating the choice of secretory mode. Syts are a family of synaptic vesicle proteins that contain two calcium-binding C 2 domains (C 2 A and C 2 B). Full fusion events led to brief "spikes" of high concentrations of released catecholamine, whereas kiss-and-run events led to more prolonged release of low concentrations of transmitter. Overexpression or pharmacological manipulations that increased Syt IV--the expression of which is increased by seizures--led to increased frequency and duration of kiss-and-run episodes. Mutational analysis of transfected Syt I and Syt IV, in combination with analysis of the calcium dependence of secretion in permeabilized cells, indicated that Syt I promoted full fusion (through its C 2 A domain) whereas Syt IV promoted kiss-and-run secretion (through its C 2 B domain). The authors speculate that prolonged release of low levels of neurotransmitter in response to increased synaptic Syt IV concentrations could desensitize postsynaptic receptors, thereby silencing the synapse and serving as a homeostatic mechanism to counteract seizure activity. C.-T. Wang, J.-C. Lu, J. Bai, P. Y. Chang, T. F. J. Martin, E. R. Chapman, M. B. Jackson, Different domains of synaptotagmin control the choice between kiss-and-run and full fusion. Nature 424 , 943-947 (2003). [Online Journal]

Effect of high concentration of glucose on dopamine release from pheochromocytoma-12 cells

To investigate the mechanism of the action of high concentration of glucose on transmitter release from neuronal cells, we examined the effect of high concentration of glucose on dopamine release from pheochromocytoma-12 (PC12) cells. When the cells were incubated with 9.0 or 13.5 mg/mL glucose (2- or 3-fold of the optimum glucose concentration for PC12 cells), dopamine release was increased in a dose-related manner. Glucose-induced increase in dopamine release was blunted by nicardipine, a Ca 2+ channel blocker. Following addition of 13.5 mg/mL glucose, intracellular Ca 2+ concentration was increased, which was eliminated by nicardipine. Administration of 9.0 or 13.5 mg/mL glucose induced membrane depolarization in a dose-related manner. Glucose-induced dopamine release was inhibited by pinacidil or diazoxide, adenosine triphosphate (ATP)-sensitive K + channel (K atp channel) openers. These results suggest that a high concentration of glucose induced ATP production, which blocked the K atp channel to induce membrane depolarization, and increased intracellular Ca 2+ concentration and dopamine release. When the cells were cultured with 9.0 or 13.5 mg/mL glucose for 7 days, high potassium chloride (KCl)-induced dopamine release and 45Ca 2+ uptake were increased. These results suggest that long-term incubation with a high concentration of glucose increased the capacity of Ca 2+ uptake to enhance depolarization-induced dopamine release from PC12 cells. These data taken together suggest that a high concentration of glucose induced activation of the Ca 2+ channel to stimulate dopamine release from PC12 cells.

Activation of tyrosine hydroxylase by intermittent hypoxia: involvement of serine phosphorylation.

Regulation of tyrosine hydroxylase (TH) by intermittent hypoxia (IH) was investigated in rat pheochromocytoma 12 (PC-12) cells by exposing them to alternating cycles of hypoxia (1% O2, 15 s) and normoxia (21% O2, 3 min) for up to 60 cycles; controls were exposed to normoxia for a similar duration. IH exposure increased dopamine content and TH activity by approximately 42 and approximately 56%, respectively. Immunoblot analysis revealed that comparable levels of TH protein were expressed in normoxic and IH cells. Removal of TH-bound catecholamines and in vitro phosphorylation of TH in cell-free extracts by the catalytic subunit of protein kinase A (PKA) increased TH activity in normoxic but not in IH cells, suggesting possible induction of TH phosphorylation and removal of endogenous inhibition of TH by IH. To assess the role of serine phosphorylation in IH-induced TH activation, TH immunoprecipitates and extracts derived from normoxic and IH cells were probed with anti-phosphoserine and anti-phospho-TH (Ser-40) antibody, respectively. Compared with normoxic cells, total serine and Ser-40-specific phosphorylation of TH were increased in IH cells. IH-induced activation of TH and the increase in total serine and Ser-40-specific phosphorylation of TH were inhibited by Ca2+/calmodulin-dependent protein kinase (CaMK) and PKA-specific inhibitors but not by inhibitors of the extracellular signal-regulated protein kinase pathway, suggesting that IH activates TH in PC-12 cells via phosphorylation of serine residues including Ser-40, in part, by CaMK and PKA. Our results also suggest that IH-induced phosphorylation of TH facilitates the removal of endogenous inhibition of TH, leading to increased synthesis of dopamine.

Hypoxic condition interferes with phosphorylation of Akt at Thr 308 in cultured rat pheochromocytoma-12 cells

Phosphorylation of Akt induced under hypoxic or ischemic conditions has been reported only for residue Ser 473. We examined whether Akt can be phosphorylated at Thr 308, another phosphorylation site on Akt, and can exhibit neuroprotective effects under conditions of hypoxia /reoxygenation, comparing pheochromocytoma-12 (PC12) cells transfected with constitutively active Akt (Myr-pCMV cells) and those transfected with pCMV vector only (pCMV cells). Expression levels of Akt phosphorylated at Ser 473 were 2.1-fold higher in Myr-pCMV cells compared with pCMV cells, before the onset of hypoxia, which were increased transiently during hypoxia, and then decreased gradually during reoxygenation. In contrast, Akt phosphorylated at Thr 308 was not detected in pCMV cells under any conditions but was expressed in Myr-pCMV cells prior to hypoxia, followed by an immediate decrease during hypoxia and a further decline during reoxygenation. However, G1-arrest of the cell cycle observed at 12 h after hypoxia in pCMV cells was prevented in Myr-pCMV cells. These findings suggest that hypoxia activates Akt by phosphorylation at Ser 473 only, which is sufficient to elicit a neuroprotective function against hypoxic neuronal damage.

Corticosterone increases serotonin type-3 receptor mRNA in rat pheochromocytoma-12 cells

Serotonin type-3 (5-HT3) receptors are cation permeable membrane receptors which are involved in modulation of calcium entry in neuronal cells. Along with other ion-channels such as the N-methyl- d-aspartate receptor, it appears to be a target for the actions of ethanol and has been the focus of considerable work in this regard. Since in animals, ethanol exposure results in elevations of corticosteroids in both acute and chronic conditions, we studied the effects of both ethanol and corticosteroid exposure on 5-HT3 gene expression in an in situ pheochromocytoma-12 (PC12) cell model. We found that ethanol exposure alone (80 mM×4 days) did not significantly alter target gene expression. Corticosterone (CORT) (50, 150, and 300 ng/ml) resulted in significant increases in 5-HT3 expression which were attenuated by mifeprestone (50 ng/ml). Ethanol in combination with CORT did not significantly alter the increase in 5- HT3 mRNA seen with CORT alone. We conclude that in PC12 cells, exposure to CORT at physiologically relevant concentrations increases 5-HT3 gene expression.

Cell density inversely regulates d- and l-aspartate levels in rat pheochromocytoma MPT1 cells

In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that d-aspartate ( d-Asp) is synthesized in rat pheochromocytoma 12 (PC12) cells. This unique amino acid is believed to act as a novel messenger in mammalian cell regulation. However, the dynamics of d-Asp homeostasis in mammalian cells is yet to be elucidated. In this communication, we demonstrate that d-Asp is also synthesized in MPT1 cells (a subclone of PC12 cells) and that the d- and l-Asp levels in cells are regulated by cell density of the culture. Our data show that d-Asp levels increase, while in contrast, l-Asp levels decrease as a function of increased cell density. Conversely, in PC12 cells, which do not express the glutamate transporter involved in the incorporation of d- and l-Asp into cells, l-Asp levels decrease upon cell density increase while d-Asp concentrations remain almost unchanged. The results indicate that the biochemical behaviors of d- and l-Asp in mammalian cells are distinct and that the cellular levels of these stereoisomers appear to be under different control mechanisms.

Methamphetamine-induced dopaminergic neurotoxicity and production of peroxynitrite are potentiated in nerve growth factor differentiated pheochromocytoma 12 cells.

Methamphetamine (METH) is a widely abused psychomotor stimulant known to cause dopaminergic neurotoxicity in rodents, nonhuman primates, and humans. METH administration selectively damages the dopaminergic nerve terminals, which is hypothesized to be due to release of dopamine from synaptic vesicles within the terminals. This process is believed to be mediated by the production of free radicals. The current study evaluates METH-induced dopaminergic toxicity in pheochromocytoma 12 (PC12) cells cultured in the presence or absence of nerve growth factor (NGF). Dopaminergic changes and the formation of 3-nitrotyrosine (3-NT), a marker for peroxynitrite production, were studied in PC12 cell cultures grown in the presence or absence of NGF after different doses of METH (100-1,000 microM). METH exposure did not cause significant alterations in cell viability and did not produce significant dopaminergic changes or 3-NT production in PC12 cells grown in NGF-negative media after 24 hours. However, cell viability of PC12 cells grown in NGF-positive media was decreased by 45%, and significant dose-dependent dopaminergic alteration and 3-NT production were observed 24 hours after exposure to METH. The current study supports the hypothesis that METH acts at the dopaminergic nerve terminals and produces dopaminergic damage by the production of free radical peroxynitrite.

Possible involvement of tetrahydrobiopterin in the trophic effect of insulin-like growth factor-1 on rat pheochromocytoma-12 (PC12) cells

Tetrahydrobiopterin (BH 4) has a trophic effect on pheochromocytoma-12 (PC12) cells such as insulin-like growth factor-1 (IGF-1). We investigated involvement of BH 4 in the trophic effect of IGF-1 on PC12 cells. IGF-1 (10–300 ng/ml) increased cellular BH 4 content in a dose-related manner. Cellular BH 4 content increased after 6–36 h incubation with IGF-1. IGF-1-induced increase in the cellular BH 4 content was blunted by 0.3 mM 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor for BH 4 synthesis. IGF-1 protected PC12 cells from the cell death induced by depletion of serum and nerve growth factor, which was attenuated by DAHP. The effects of IGF-1 on the cellular BH 4 content and cell viability were eliminated by 0.2 μM wortmannin. These results suggest that BH 4 is involved in the trophic effect of IGF-1 on PC12 cells and that the effect of IGF-1 on BH 4 synthesis is mediated by phosphatidylinositol 3-kinase.