Nerve Growth Factor and Gangliosides Stimulate the Release of Glycoproteins from PC 12 Pheochromocytoma Cells

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.     

The Action of Adenosine Analogs on PC12 Cells

Abstract: PC12 cells, a nerve growth factor–responsive clone of rat pheochromocytoma, contain a membrane–bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase–stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5'- N -ethyIcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine–evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non–histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.     

Vesamicol Binding to Subcellular Membranes That Are Distinct from Catecholaminergic Vesicles in PC 12 Cells

We have examined PC12 cells for the localization of binding sites for vesamicol [l-2-(4-phenylpiperidino) cyclohexanol], a compound that has previously been shown to bind to cholinergic vesicles and to inhibit the uptake of acetylcholine. Initial studies presented in this article demonstrate the existence of a specific, saturable vesamicol binding site in PC12 cells. Subsequent experiments show that these binding sites reside in a membrane population that is distinct from catecholamine-containing compartments with respect to density and antigenic composition. In particular, vesamicol binding compartments have a lower density than catecholaminergic vesicles and, unlike these latter vesicles, do not appear to contain the vesicle-specific proteins synaptophysin and SV2 as part of the same membrane. These results suggest that vesicular transport proteins for acetylcholine and catecholamines are differentially sorted to distinct membrane compartments in PC12 cells.     

Monoamine Oxidase (MAO)-A but Not MAO-B Inhibitors Potentiate Tyramine-Induced Catecholamine Release from PC12 Cells

The previous report that PC12 (pheochromocytoma) cells have a K(+)-induced, as well as a tyramine-induced, catecholamine release mechanism has been confirmed. Selective monoamine oxidase (MAO)-A (clorgyline and moclobemide) and not MAO-B inhibitors (l-deprenyl, AGN 1135, and Ro 16-6491) potentiate the catecholamine-releasing action of tyramine significantly more than that of K+. The potentiation of tyramine-induced [3H]noradrenaline release from PC12 cells by MAO-A inhibitors has been linked to the presence of MAO-A in these cells, for which tyramine and noradrenaline are substrates. In the above respects, it is the PC12 cell that resembles more closely the peripheral adrenergic neuron, rather than the chromaffin cell, which is endowed with MAO-B and lacks the tyramine-releasable pool of catecholamines.     

Antisense GAP-43 Inhibits the Evoked Release of Dopamine from PC12 Cells

Abstract: To investigate the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, F1, P-57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP-43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP-43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP-43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K⁺ (56 m M ) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP-43 RNA in PC12 cells leads to a decrease in GAP-43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP-43 in calcium-dependent neurotransmitter release.     

Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.     

Nerve Growth Factor Potentiates Bradykinin-Induced Calcium Influx and Release in PC12 Cells

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PC12 cells. Short-term (1 h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10-fold, whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1,4,5-trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.     

Release of the NILE and Other Glycoproteins from Cultured PC 12 Rat Pheochromocytoma Cells and Sympathetic Neurons

Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4-8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr less than or equal to 230,000. Another major component of apparent Mr = 55,000 as well as minor components of apparent Mr less than or equal to 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr less than or equal to 210,000 was released only by the sympathetic neurons. The soluble released Mr less than or equal to 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF-inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane-derived NILE GP. At least several of the other released GPs appeared to be derived from membrane-bound components with which they share immuno-crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS-polyacrylamide gels than their apparent membrane-bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.     

Digitonin-Permeabilized Cells Are Exocytosis Competent

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.     

Regulation of Depolarization-Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP-Dependent Enhancement of Release from PC12 Cells

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.     

Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

Differential Ras-Dependence of Gene Induction by Nerve Growth Factor and Second Messenger Analogs in PC12 Cells

Induction of neurite formation by nerve growth factor (NGF) in PC12 pheochromocytoma cells can be efficiently inhibited by expressing a dominant negative mutant form of the small guanine nucleotide binding Ha-Ras protein in these cells. The block in NGF-induced neuritogenesis caused by inhibition of endogenous Ras proteins was found to be partially relieved by simultaneous stimulation of cAMP- or Ca⁺⁺-dependent signaling pathways. Since expression of certain genes is believed to be involved in NGF-signaling leading to morphological differentiation, we decided to study the combined effects of NGF and second messenger analogs on gene expression in PC12 cell lines expressing different levels of the interfering Ras protein. We found NGF-second messenger combinations that induced normal c-fos, zif268 and nur77 early-response gene expression without neuritogenesis, and, conversely, cell lines in which certain combination treatments caused partial neuronal differentiation in the absence of substantial activation of these genes. Similarly, neurite outgrowth induced by combination treatments does not seem to require the activation of the late-response transin gene. Our results thus suggest a lack of strong correlation between NGF-stimulated early- and secondary-response gene induction and morphological differentiation.     

Aluminum-induced apoptosis in PC12D Cells

The addition of aluminum-maltol complex to PC12D cells induced a time-dependent and concentration-dependent growth inhibition as well as cell death, whereas aluminum chloride or maltol alone did not affect the viability of PC12D cells. Apoptosis of differentiated PC12D cells was assessed by using terminal deoxynucleotidyltransferase-mediated 2-deoxyuridine-5-triphosphate nick end labeling (TUNEL) technique to detect DNA strand breaks in situ. The number of TUNEL-positive cells treated with aluminum-maltol increased with time in the treatment cultures. The ability of aluminum ion to elevate intracellular reactive oxygen species was determined by fluorescence in PC12D cells loaded with the oxidant-sensitive dye 2,7-dichlorofluorescin diacetate. Aluminum ion incorporated to PC12D cells causes apoptotic cell death by enhancing the generation of reactive oxygen species.     

The Latency of Exocytosis Varies with the Mechanism of Stimulated Release in PC12 Cells

Abstract: To compare the time course of different mechanisms of chemically stimulated release, amperometric detection of dopamine was carried out at single PC12 cells. The rapid response of carbon fiber microelectrodes allowed the detection of single exocytotic events, thus providing time-resolved information about the dynamics of stimulated release, in particular the latency between the stimulation of a cell and the secretion of catecholamines. On rapid depolarization of the cell membrane caused by application of 105 m M K⁺, almost immediate (6 ± 1 s) release of dopamine was observed. Stimulation with 1 m M nicotine, involving the stimulant binding to a ligand-gated ion channel, resulted in a short (37 ± 5 s) delay between stimulation and secretion. Application of 1 m M muscarine to the cells caused a long (103 ± 11 s) latency before exocytosis was detected. A biphasic response that appeared to be similar to a combination of nicotine- and muscarine-stimulated release was observed when cells were stimulated with 10 m M acetylcholine. Thus, it appears that the dynamics of stimulated release at single PC12 cells is significantly affected by the mechanism leading to exocytosis.     

三七素对谷氨酸损伤的PC12 细胞的影响

目的:研究三七素对谷氨酸损伤的PC12细胞的影响。方法:用谷氨酸复制体外培养的PC12细胞损伤模型,采用MTT法、Hoechst33342/PI双重染色法分别研究高、低剂量三七素对谷氨酸所致的PC12细胞损伤的影响。结果:谷氨酸刺激后,PC12细胞存活率较对照组显著降低(P<0.05)、凋亡显著增强(P<0.05)。给予高剂量三七素可加重谷氨酸引起的PC12增殖力降低、凋亡增强。但低剂量三七素干预后,细胞存活率较模型组显著升高(P<0.05),凋亡较模型组明显降低(P<0.05)。结论:高剂量三七素可加重谷氨酸对PC12细胞的损伤,但低剂量三七素可显著减轻谷氨酸对PC12细胞的损伤,但具体机制尚有待于进一步研究。     

Structure of Catecholamine Secretory Vesicles from PC12 Cells

Catecholamine secretory organelles were partially purified from PC12 cells. Measurement of the sedimentation coefficient (540S in 0.32 M sucrose), density in an isoosmotic gradient (1.139 g/cm), and density in an isoosmotic gradient using D2O as a solvent (1.205 g/cm3) have allowed us to calculate the molecular weight (1.17 X 10(9) daltons), radius (74 nm), and water content (62% vol/vol) of the secretory vesicle. The vesicle appears to contain ATP, but the molar ratio of 3,4-dihydroxyphenylethylamine (dopamine) to ATP in the particles is high (16.5) and the ATP was frequently asymmetrically distributed in the vesicle fraction. The particle behaves like a true secretory particle in that the dopamine content of the particle is increased by pargyline, diminished by depolarization, and abolished by reserpine. Sequential purification of PC12 lysates on controlled pore glass columns and isoosmotic Ficoll gradients produced a 20-30-fold purification, but this enrichment is not sufficient to produce a homogeneous population of vesicles. An 82,000-dalton protein copurifies with secretory granules and appears to be the major secreted protein. At this stage of purification this single protein makes up about 30% of the protein in the vesicle-containing fractions and so the vesicles must be approaching homogeneity.     

Paraquat-Induced Cell Death in PC12 Cells

Paraquat was taken up by PC12 cells in a carrier-mediated, saturable manner. When PC12 cells were permeabilized with digitonin (50 g/ml) lipid peroxidation was observed after paraquat treatment in the presence of NADPH and chelated iron. The fact that lipid peroxidation preceded the appearance of LDH release provides positive evidence that lipid peroxidation may be one of the important factors leading to cytotoxicity of cells. Furthermore, the fact that addition of superoxide dismutase, catalase and promethazine efficiently blocked the malondialdehyde formation and attenuated the cell death indicated the involvement of reactive oxygen radicals in mediating the cytotoxicity induced by paraquat. Taken together the results present in vitro evidence that neurotoxicity of paraquat may be a consequence of cellular lipid peroxidation, which leads to cell death and may have great implications in assessing the risk of exposure to paraquat in Parkinson's disease.