PC12D Cells Research Articles

Overexpression of V-1 prevents nitric oxide-induced cell death: involvement of enhanced tetrahydrobiopterin biosynthesis.

Previously we reported that the synthesis of catecholamines, dopamine, and noradrenaline was enhanced by overexpression of V-1 protein, a neuronal protein active in the initial stage of development of the rat cerebellum, in the neuronal cell line PC12D, a model of dopamine cells (Yamakuni et al. [1998] J. Biol. Chem. 273:27051-27054). To investigate the physiological role of this protein, we examined the effect of V-1 overexpression on cell toxicity induced by nitric oxide (NO) used at low concentrations. Two clones of PC12D cells overexpressing V-1, transfectants termed V1-46 and V1-69, were significantly more resistant to NOR3 (an NO donor) but not to etoposide (an inhibitor of topoisomerase II)-induced apoptotic cell death than the control cells (termed C-7 and C-9) that had been transfected with the vector alone. The addition of L-DOPA, dopamine, or noradrenaline to the medium did not abolish NOR3-induced cell death in PC12D cells. Moreover, pretreatment of V1-46 and V1-69 cells with L-alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase, to inhibit catecholamine biosynthesis did not affect the resistance to NO toxicity. These results indicate that the catecholamine levels increased by V-1 overexpression did not produce the protection against NOR3-induced toxicity. We further showed that overexpression of V-1 enhanced the synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). In addition, pretreatment with BH(4) or with sepiapterin, which is converted to BH(4) intracellularly, significantly protected PC12D cells in a dose-dependent manner. The increased BH(4) synthesis by V-1 overexpression was dose dependently inhibited by pretreatment with diaminohydroxypyrimidine (DAHP), an inhibitor of GTP-cyclohydrolase I, which is the rate-limiting enzyme for the biosynthesis of BH(4), concomitantly with the loss of protective effect afforded by V-1 overexpression. Furthermore, the addition of BH(4) or sepiapterin to DAHP-pretreated V146 and V1-69 cells restored cell viability. Taken together, these results indicate that V1 protein plays an important role in protection against cell death induced by NO at low levels by promoting the synthesis of BH(4). Moreover, these findings suggest the up-regulation of V1 expression as a possible therapeutic target for protection against the insult of NO-induced oxidative stress.

Sterol and triterpenoid constituents of Verbena littoralis with NGF-potentiating activity.

Two new sterols, stigmast-5-ene 3beta,4beta,7alpha,22alpha-tetraol (1) and stigmast-5-ene 3beta,7alpha,22alpha-triol (2), were isolated from the aerial parts of a Paraguayan medicinal plant, Verbena littoralis, together with the known compounds ursolic acid (3) and oleanolic acid (4). The structures of 1 and 2 were elucidated by spectroscopic analyses. Compounds 2-4 showed an enhancing activity of nerve growth factor (NGF)-mediated neurite outgrowth in PC12D cells.

Nardosinone enhances nerve growth factor-induced neurite outgrowth in a mitogen-activated protein kinase- and protein kinase C-dependent manner in PC12D cells.

The mechanism to enhance nerve growth factor (NGF, 2 ng/ml)-induced neurite outgrowth from PC12D cells by nardosinone isolated from Nardostachys chinensis was examined. It was shown that the potentiation of the NGF-induced neurite outgrowth by nardosinone was mitogen-activated protein (MAP) kinase-dependent, but was not accompanied by stimulation of NGF-induced increase in MAP kinase phosphorylation. Furthermore, this augmentation of NGF-induced neurite outgrowth was abolished by GF109203X, a protein kinase C (PKC) inhibitor. These results suggest that the enhancement of NGF-induced neurite outgrowth from PC12D cells by nardosinone involves activation of a down-stream step of the MAP kinase-dependent cascade of NGF coupled with PKC.

A new iridoid glycoside with nerve growth factor-potentiating activity, gelsemiol 6'-trans-caffeoyl-1-glucoside, from Verbena littoralis.

A new iridoid glycoside, gelsemiol 6'-trans-caffeoyl-1-glucoside (1), was isolated from Verbena littoralis, together with four known phenylethanoid glycosides, acteoside (2), 2'-acetylacteoside (3), jionoside (4), and isoverbascoside (5). Their structures were elucidated by spectral data. Compound 1 showed weak enhancement of nerve growth factor (NGF)-mediated neurite outgrowth from PC12D cells.

Prenylated xanthones from Garcinia xanthochymus.

A new prenylated xanthone, 1,3,5,6-tetrahydroxy-4,7,8-tri(3-methyl-2-butenyl)xanthone (1), was isolated from the wood of Garcinia xanthochymus together with a known xanthone, garciniaxanthone E (2). Their structures were determined by spectroscopic analysis. Compounds 1 (3 microM) and 2 (10 microM) elicited marked enhancement of nerve growth factor-mediated neurite outgrowth in PC12D cells.

Littorachalcone, a new enhancer of NGF-mediated neurite outgrowth, from Verbena littoralis.

A new dihydrochalcone dimer, 2',4',3",2"',4"'-pentahydroxy-4-O-4"-tetrahydrobichalcone, given the name littorachalcone, was isolated from the aerial parts of Verbena littoralis H. B. K. along with two known flavonoids 4'-hydroxywogonin and 8,3'-dimethoxy-5,7,4'-trihydroxyflavone. Littorachalcone caused a significant enhancement of nerve growth factor-mediated neurite outgrowth from PC12D cells.

神経成長因子の濃度勾配刺激によるPC12D細胞のlamllipodia伸展収縮運動と神経様突起形成

神経成長因子の濃度勾配刺激によるPC12D細胞のlamllipodia伸展収縮運動と神経様突起形成

Expression of V-1, a novel catecholamine biosynthesis regulatory protein, is enhanced by hypertension in atrial myocytes of Dahl salt-sensitive rats

V-1 positively controls catecholamine synthetic gene transcription to promote catecholamine production in PC12D cells. In this study, immunohistochemical analysis revealed that in Wistar rats, V-1 immunoreactivity was localized not only in sympathetic axons but also in the cytoplasm of cardiomyocytes, and that the immunoreactivity in atrial myocytes was more intense than that in ventricular myocytes. Western blot analysis also showed that V-1 expression level in the atrium was higher than that in the ventricle of Wistar rat hearts. When Dahl salt-sensitive (DS) rats were fed an 8% NaCl diet after the age of 6 weeks, blood pressure was raised 230 mm Hg at 18 weeks. V-1 expression was shown to be increased in the atrial myocytes of these DS rats, but not in the sympathetic axons, when assayed by immunohistochemistry. These results suggest that in normotensive rats, V-1 is preferentially expressed in the cytoplasm of cardiomyocytes in the atrium rather than in the ventricle. It is also suggested that V-1 expression is increased by hypertension in DS rat atrium.

Identification of ATF-2 as a Transcriptional Regulator for the Tyrosine Hydroxylase Gene

Transcriptional regulation of catecholamine-synthesizing genes is important for the determination of neurotransmitters during brain development. We found that three catecholamine-synthesizing genes were transcriptionally up-regulated in cloned PC12D cells overexpressing V-1, a protein that is highly expressed during postnatal brain development (1). To reveal the molecular mechanism to regulate the expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme for catecholamine biosynthesis, we analyzed the transcription factors responsible for TH induction in the V-1 clonal cells. First, by using reporter constructs, we found that the transcription mediated by cAMP-responsive element (CRE) was selectively enhanced in the V-1 cells, and TH promoter activity was totally dependent on the CRE in the promoter region of the TH gene. Next, immunoblot analyses and a transactivation assay using a GAL4 reporter system revealed that ATF-2, but not cAMP-responsive element-binding protein (CREB), was highly phosphorylated and activated in the V-1 cells, while both CREB and ATF-2 were bound to the TH-CRE. Finally, the enhanced TH promoter activity was competitively attenuated by expression of a plasmid containing the ATF-2 transactivation domain. These data demonstrated that activation of ATF-2 resulted in the increased transcription of the TH gene and suggest that ATF-2 may be deeply involved in the transcriptional regulation of catecholamine-synthesizing genes during neural development.

V-1, a catecholamine biosynthesis regulatory protein, positively controls catecholamine secretion in PC12D cells

Stably transfected PC12D cell lines overexpressing a catecholamine biosynthesis regulatory protein, V-1, were used to examine the functional role of V-1 in catecholamine secretion. High K +-induced dopamine secretion in V-1 overexpressing clones was shown to be markedly potentiated compared with control clones carried with a vector alone. As assayed intracellular calcium concentration ([Ca 2+] i) using fura-PE3, V-1 overexpression was observed to enhance high K +-elicited [Ca 2+] i elevation. Electron microscopic analysis revealed an increase in dense-cored vesicle formation by V-1 overexpression. These results suggest that the enhancement of high K +-induced dopamine secretion by V-1 overexpression results from the potentiation of high K +-induced [Ca 2+] i elevation and the increase in the number of dense-cored vesicles.

Utilization of a copper-catalyzed diaryl ether synthesis for the preparation of verbenachalcone

2-Hydroxy-2′-methoxydiphenyl ethers were efficiently assembled utilizing a catalytic copper mediated coupling of 2-benzyloxybromobenzene derivatives and 2-methoxyphenol derivatives in the presence of cesium carbonate in pyridine followed by debenzylation. Utilization of this method allowed for a concise synthesis of verbenachalcone, a compound reported to enhance nerve growth factor's ability to stimulate neurite outgrowths in PC12D cells. Initial SAR data is also presented.

Glucocorticoid inhibits expression of V-1, a catecholamine biosynthesis regulatory protein, in cultured adrenal medullary cells

V-1 acts as a positive and coordinate regulator of gene expression of catecholamine biosynthetic enzymes in PC12D cells. The present study was conducted to investigate the mechanism controlling expression of V-1 in the adrenal gland. Immunohistochemical analysis demonstrated that noradrenergic chromaffin cells more highly expressed V-1 than adrenergic chromaffin cells preferentially expressing the glucocorticoid receptor in rat adrenal glands. Western blotting showed that in cultured bovine adrenal medullary cells, dexamethasone, a synthetic glucocorticoid, inhibited expression of V-1, and that this inhibition was prevented by RU-486, a glucocorticoid receptor antagonist. These results suggest that adrenal expression of V-1 is differentially controlled by glucocorticoids through the specific receptor, and that thereby V-1 regulates catecholamine biosynthesis in a catecholaminergic phenotype-dependent manner.

Picrosides I and II, selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 μM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 μM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 μM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.

Enhanced expression of GTP cyclohydrolase I in V-1-overexpressing PC12D cells

Three of the catecholamine-synthesizing enzymes, i.e., tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase, and dopamine β-hydroxylase, were earlier shown to be up-regulated in cloned PC12D cells overexpressing V-1, a cdc10/SWI6 motif-containing protein. GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH 4), known as an essential cofactor for TH; and here we found the increased expression of GCH in V-1-overexpressing clones. Both GCH activity and total biopterin content were highly increased in the V-1 clones; whereas the activity of sepiapterin reductase, enzyme in the final step of the BH 4 biosynthesis, was not altered. Biochemical analyses revealed increased levels of GCH protein, mRNA, and transcription in the V-1 clones. Promoter analysis showed increased reporter activity in the construct with 150 bp of the promoter region of the human GCH gene, suggesting the involvement of cAMP-responsive element-mediated transcriptional regulation.

Effect of magnetic field exposure on calcium channel currents using patch clamp technique.

Calcium influxes through the membrane of PC-12D cells were measured under exposure to DC biased AC magnetic fields in resonant conditions of the ion cyclotron and the ion parametric resonance hypotheses and compared with influxes in cells without exposure to the magnetic field. After cancellation of the geomagnetic field, the cells were exposed to the horizontal fields generated by a current sheet, a planar sheet of conductor which generated a satisfactorily homogeneous horizontal magnetic field on the stage of a microscope without hindering treatment of a cell under observation. At or near any resonant conditions, no change in calcium influx could be detected under standard patch clamp conditions.

Stimulation of neurite outgrowth in PC12D cells by ELF magnetic field and suppression by melatonin

PC12D cells pertain to the subline of rat pheochromocytoma cells and are differentiated into nerve-like cells by NGF (nerve growth factor) or forskolin. In order to clarify the effect of magnetic fields at extremely low frequency (ELF) on nervous system in vitro, we observed neurite outgrowth in PC12D, which was induced for 22 h culture by 10 μM forskolin in both a pair of Helmholtz coils and a magnetically shielded box. Under the magnetic fields of 33.3 μT in vertical at 60 Hz and of geomagnetism, the neurite factor F was increased 1.3 times greater than that in the shielded box; F=( f− f 0)/( f R− f 0), where f= n +/ n T, n + is the number of neurite-bearing cells for the total number n T of cells ( n T∼180) counted under microscope and the subscripts R and 0 denote f's of cells with 10 μM and no forskolin, respectively. Furthermore we observed the influence of melatonin, since melatonin is expected to protect the electromagnetic damage to living organisms. F decreased at the three concentrations of 0.11, 5.3 and 260 nM melatonin significantly as compared with that at no melatonin under the magnetic fields, although F was not significantly varied under no magnetic fields except that at 260 nM melatonin. It is concluded that ELF magnetic field stimulated neurite outgrowth in PC12D cells induced by forskolin and melatonin suppressed its effect at low concentration corresponding to physiological level of melatonin. It is discussed on the relation between neurite outgrowth and Ca 2+ influx through membrane.

Circular Nuclear Alignment in Multinucleate PC12D Cells Produced by Cell Fusion with Polyethylene Glycol.

PC12D cells, a subline of PC12 cells, extend neurites within a few hours in response to nerve growth factor (NGF). Multinucleate PC12D cells were produced by cell fusion with polyethylene glycol (PEG). The syncytia extended large neurites following treatment with NGF. In the syncytia, nuclei were distributed in a circular alignment. Many nuclear rings were found in the culture dish 1 day after treatment with PEG, although they were sometimes seen in syncytia within several hours. Colchicine prevented this alignment or disrupted the clusters when added subsequent to their formation. Cytochalasin B did not prevent the formation of these rings, nor affect the alignment of nuclei. Immnofluorescent staining of γ-tubulin, an integral protein of the centrosome, revealed intense dot-like structures in the center of nuclear rings in syncytia. Twice as many dots, or centrioles, as nuclei were present in each syncytium. However, most microtubules were localized around the nuclei and did not appear to extend from the cluster of centrioles. Fluorescent labeling of mitochondria showed them to mainly be localized inside the nuclear rings. Meanwhile, immunofluorescently stained endoplasmic reticulum was mainly localized along with the circular alignment of nuclei. Therefore, not only nuclei but various cell organelles are situated in each characteristic location in the syncytia. These data suggest that microtubules are involved in the migration and alignment of nuclei and presumably of cell organelles in the syncytia.

1N0945 PC12D細胞のlamellipodia伸展収縮とNGF刺激による神経様突起形成(12.細胞生物学的課題,一般演題,日本生物物理学会第40回年会)

1N0945 PC12D細胞のlamellipodia伸展収縮とNGF刺激による神経様突起形成(12.細胞生物学的課題,一般演題,日本生物物理学会第40回年会)

Neurite outgrowth promoting activity of marine algae from Japan against rat adrenal medulla pheochromocytoma cell line, PC12D.

We screened for biological activity which induces neurite outgrowth in vitro from 300 species of marine algae from along the Japan coast for possible use as a treatment for the lack of neurotrophic factor which is considered to be a cause of Alzheimer's disease. In this study, we evaluated the neurite outgrowth promoting activity in a rat adrenal medulla pheochromocytoma cell line, PC12D, using a low level of NGF (nerve growth factor). Although most of the samples had no activity, MeOH extract from a brown alga, Sargassum macrocarpum and PBS extract from a red alga, Jania adharens, exhibited neurite outgrowth promoting activity and induced neuron specific dendrites and axons from the surfaces of PC12D cells. The active substance present in S. macrocarpumseemed to be lipid and heat stable with molecular weight of around 500 to 1000. These results suggest that marine algae may constitute a good source for development of promising novel agents with neurotrophic activity in brain nerve systems for future use in treatment of Alzheimer's disease.

Novel effect of vitamin K 1 (phylloquinone) and vitamin K 2 (menaquinone) on promoting nerve growth factor-mediated neurite outgrowth from PC12D cells

The nerve growth factor (NGF)-potentiating effect of K vitamins on PC12D cells was investigated. Treatment of PC12D cells with vitamin K 1 or K 2 in the presence of NGF significantly enhanced the proportion of neurite-bearing cells and acetylcholinesterase activity compared with NGF treatment alone. The K vitamins-enhanced neurite outgrowth on PC12D cells was completely blocked by a protein kinase A (PKA) inhibitor or mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059, whereas a protein kinase C inhibitor chelerythrine chloride did not significantly inhibit the enhancing effect of the K vitamins. These results suggest that the K vitamins enhance neurite outgrowth via the activation of PKA and MAPK-mediated signaling pathways in PC12D cells.