Rat Adrenal Pheochromocytoma PC12 Cells Research Articles

177 Differentiation-Dependant Sensitivity Of Neuronal Precursor Cells Towards Oxidative Stress

Background: Apoptosis is an endogenous cell suicide mechanism triggered in response to biological factors and genotoxic stimuli often resulting from oxidative stress. Neuronal apoptosis especially in the developing brain may cause long-term brain dysfunction. Cognitve deficits in the later childhood are frequent in preterm infants. Preterm infants are exposed to high oxygen due to their premature lungs. At the same time their nutritional regimens are deficient in antioxidant precursors. In cell cultures a relation between oxidative stress and cell death could be found. This study tested the hypothesis that differentiated rat adrenal medullary pheochromocytoma PC12 cells are less sensitive to hyperoxia than undifferentiated cells.Methods: PC12 cells differentiated with nerve growth factor protein (NGF) and undifferentiated cells were exposed to an atmosphere of 80% oxygen and treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. Control cells did not receive BSO. Cell viability was tested by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide). Apoptosis was measured by flow cytometry (annexin/ propidiumjodid, caspase-3-activity).Results: Undifferentiated cells treated with BSO exposed to hyperoxia showed an increase in apoptosis (cytometry) and a significant decrease in viability (BSO+normoxia 81±5,1%, hyperoxia 95±3,1%, BSO+hyperoxia 22±3,0%, means ± S.E.M. for 4– 6 experiments, p< 0,05, cell viability assay). Differentiated cells showed reduced sensibility towards BSO and hyperoxia (BSO+normoxia 101±3,3%, hyperoxia 97±1,9%, BSO+hyperoxia 87,6±2,5%, cell viability assay). BSO alone did not induce apoptosis.Conclusion: We conclude that exposure to high oxygen in combination with limited antioxidant protection is responsible for increased cell death via apoptosis in undifferentiated neurons. This finding may contribute to longterm cognitive deficits in preterm infants exposed to high oxygen.

Adaptive changes in the expression of nuclear and mitochondrial encoded subunits of cytochrome c oxidase and the catalytic activity during hypoxia.

The effects of physiologically relevant hypoxia on the catalytic activity of cytochrome c oxidase (CytOX), mitochondrial gene expression, and both nuclear and mitochondrial encoded CytOX mRNA levels were investigated in murine monocyte macrophages, mouse C2C12 skeletal myocytes and rat adrenal pheochromocytoma PC12 cells. Our results suggest a coordinated down regulation of mitochondrial genome-coded CytOX I and II and nuclear genome-coded CytOX IV and Vb mRNAs during hypoxia. Hypoxia also caused a severe decrease in mitochondrial transcription rates, and associated decrease in mitochondrial transcription factor A. The enzyme from hypoxia exposed cells exhibited altered subunit content as revealed by blue native gel electrophoresis. There was a generalized decline in mitochondrial function that led to a decrease in total cellular heme and ATP pools. We also observed a decrease in mitochondrial heme aa3 content and decreased levels of CytOX subunit I, IV and Vb, though the catalytic efficiency of the enzyme (TN for cytochrome c oxidase) remained nearly the same. Increased glycolytic flux and alterations in the kinetic characteristics of the CytOX might be the two mechanisms by which hypoxic cells maintain adequate ATP levels to sustain life processes. Reoxygenation nearly completely reversed hypoxia-mediated changes in CytOX mRNA contents, rate of mitochondrial transcription, and the catalytic activity of CytOX enzyme. Our results show adaptive changes in CytOX structure and activity during physiological hypoxia.

Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts

The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain-specific cannabinoid receptor, while in mouse peritoneal cells, Δ 9-tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1–1 μM) was found to stimulate the phospholipase D-mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D-activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 μM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system.

Biosynthesis and degradation of bioactive fatty acid amides in human breast cancer and rat pheochromocytoma cells--implications for cell proliferation and differentiation.

The endogenous cannabinoid, anandamide (arachidonoylethanolamide), and the sleep-inducing factor, oleamide (cis-9-octadecenoamide), represent two classes of long-chain fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human breast carcinoma EFM-19 cells and rat adrenal pheochromocytoma PC-12 cells, together with the enzyme responsible for their degradation, fatty acid amide hydrolase, and the proposed biosynthetic precursors for arachidonoylethanolamide and related acylethanolamides, the N-acyl-phosphatidylethanolamines. Lipids extracted from cells labelled with [14C]ethanolamine contained radioactive compounds with the same chromatographic behaviour as arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with ionomycin in EFM-19 cells or two-week treatment with the nerve growth factor in PC-12 cells. The chemical nature of arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of oleamide. The latter compound, which does not activate the central CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than arachidonoylethanolamide (IC50 = 11.3 microM for oleamide and 2.1 microM for arachidonoylethanolamide), while at a low, inactive dose it potentiated an arachidonoylethanolamide cytostatic effect. The CB1 receptor selective antagonist SR 141716A (0.5 microM) reversed the effect of both arachidonoylethanolamide and oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [14C]arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for fatty acid amide hydrolase. This enzyme was inhibited by oleamide in both intact cells and cell-free preparations. The presence of transcripts of fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total RNA. The rate of [14C]arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of arachidonoylethanolamide enzymatic hydrolysis and the amounts of the fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with nerve growth factor and subsequent transformation of PC-12 cells into neuron-like cells. These data show for the first time that: (a) induction by nerve growth factor of a sympathetic neuronal phenotype in PC-12 cells has no effect on arachidonoylethanolamide/oleamide metabolism, (b) arachidonoylethanolamide and oleamide are autacoid suppressors of human breast cancer cell proliferation. Moreover these data lend conclusive support to the previous hypothesis that oleamide may act as an enhancer of arachidonoylethanolamide actions through competitive inhibition of its degradation.

Human Apolipoprotein E Receptor 2: A NOVEL LIPOPROTEIN RECEPTOR OF THE LOW DENSITY LIPOPROTEIN RECEPTOR FAMILY PREDOMINANTLY EXPRESSED IN BRAIN

Isolation and characterization of a human cDNA demonstrated a novel lipoprotein receptor designated apolipoprotein E receptor 2 (apoER2). The new receptor consists of five functional domains resembling the low density lipoprotein (LDL) and very low density lipoprotein (VLDL) receptors. LDL receptor deficient Chinese hamster ovary cells expressing human apoER2 bound apoE rich beta-migrating VLDL with high affinity and internalized. LDL was bound with much lower affinity to these cells. The 4.5- and 8.5-kb mRNAs for the receptor were most highly expressed in human brain and placenta. In rabbit tissues, multiple species of the mRNA with 4, 4.5, 5.5, 8.5, and 11 kb were detected most intensely in brain and testis and, to a much lesser extent, in ovary, but were undetectable in other tissues. In rat adrenal pheochromocytoma PC12 cells, the receptor mRNA was induced by treatment of the cells with nerve growth factor. The receptor transcripts were detectable most intensely in the cerebellar cortex, choroid plexus, ependyma, hippocampus, olfactory bulb and, to a much lesser extent, in the cerebral cortex as revealed by in situ hybridization histochemistry. In the cerebellar cortex, the receptor transcripts were densely deposited in Purkinje cell somata.

Treatment of PC12 cells by nerve growth factor, dexamethasone, and forskolin. Effects on cell morphology and expression of neurotensin and tyrosine hydroxylase.

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.

K + ionophores inhibit nerve growth factor-induced neuronal differentiation in rat adrenal pheochromocytoma PC12 cells

Incubation with a K +/H + ionophore nigericin attenuated the nerve growth factor (NGF)-induced neurite outgrowth in rat pheochromocytoma PC12 cells. However, a Na +/H + ionophore monensin and a Ca 2+ ionophore A23187 did not inhibited the neurite outgrowth. Nigericin also inhibited the NGF-caused induction of acetylcholinesterase and suppression of cell proliferation. These changes were dependent on the amount of the ionophore added to the culture. In addition, a distinct K + ionophore, valinomycin, similarly inhibited the NGF-induced neuronal differentiation. These results suggest the presence of the K + ionophore-sensitive mechanism in the NGF-induced differentiation system in PC12 cells.

NICER elements: a family of nerve growth factor-inducible cAMP-extinguishable retrovirus-like elements.

We have shown previously that the transcription of the gene designated d5 is induced by nerve growth factor (NGF) in rat adrenal pheochromocytoma PC-12 cells and that this NGF induction is repressed by cAMP. In this paper we demonstrate that d5 is a member of a gene family that contains several hundred members, which is closely related to retroviruses and retrotransposons, as demonstrated by the following observations: (i) the original d5 cDNA hybridized to numerous restriction fragments in genomic DNA; (ii) d5 cDNA hybridized to genomic clones with various intensities, and genomic clones can be isolated with a frequency suggesting that this family includes several hundred members; and (iii) there were minor sequence variations in four independently isolated cDNA clones that were homologous to d5 cDNA. Primer extension studies show that initiation of the 5.7-kilobase d5 mRNA(s) occurs at a unique site relative to a synthetic primer. The 5' end of the cDNA sequence was homologous to Rasheed rat sarcoma virus; and a genomic clone contained several elements that are typical of a long terminal repeat (LTR), including a CCAAT box, a TATA box, a primer binding site, a poly(A) addition signal, and a poly(A) addition site. Furthermore, there is a LTR at the 3' end of at least one of the genes in this family, and there appeared to be a four-base duplication at the probable site of integration into host DNA. Since several members of this family retain responses to NGF and cAMP, we conclude that the regulatory elements present in the LTR have been conserved in many members of this family. We have named this family of genes the NICER elements because they are a family of NGF-inducible cAMP-extinguishable retrovirus-like elements.

Electron Microscopic Localization of Acridine Orange Chromatin Interaction Products in Rat Pheochromocytoma PC12 Cells

The purpose of the present study is to examine the distribution pattern of acridine orange (AO) binding to DNA in rat adrenal pheochromocytoma PC12 cells that respond to nerve growth factor (NGF). PC12 cells were incubated in a medium containing 100 ng/ml of NGF for 1-14 days for AO ultracytochemistry. Electron microscopic studies revealed that AO binds to DNA exclusively within the euchromatin portion of the cell nucleus. About 35% of the untreated PC12 cells showed characteristic electron-dense interaction products within the nuclei. The average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area were 45 and 1.6, respectively. In the presence of NGF, cell proliferation was suppressed. The cells gradually extend neurite-like cell processes. Mean 3H-labeling index was 40.0 +/- 2.2% in untreated cells and 13.0 +/- 2.3% in PC12 cells with NGF for 6 days after incubation for 30 min. With increasing the cellular differentiation percentages of AO positive cells and average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area showed a progressive decrease. The results of the present and previous studies suggest that AO chromatin interaction products may be indicative of cell proliferation and differentiation.