Affinity labeling of a nerve growth factor receptor component on rat pheochromocytoma (PC12) cells

Clonal PC12 rat pheochromocytoma cells were sequentially incubated with 125I-labeled nerve growth factor and the photoreactive bifunctional agent hydroxysuccinimidyl-p-azidobenzoate. This treatment effected the crosslinking of 125I nerve growth factor to a PC12 cell component that exhibits an apparent Mr = 148 000-158 000, and consists of a single polypeptide chain with internal disulfide bonds. The amount of label associated with this Mr = 148 000-158 000 species was proportional to the degree of occupancy of nerve growth factor receptors by 125I-labeled nerve growth factor. Affinity labeling of this species was inhibited by the presence of 0.2 microM unlabeled nerve growth factor during incubation of PC12 cells with 125I nerve growth factor. In membranes prepared from PC12 cells hydroxysuccinimidyl-p-azidobenzoate effected the crosslinking of 125I-labeled nerve growth factor to an Mr = 120 000-130 000 species but not to the Mr = 148 000-158 000 component observed in intact cells. The kinetics of 125I nerve growth factor affinity labeling of the Mr = 148 000-158 000 species closely paralleled the time-course of 125I nerve growth factor association to two kinetically distinct forms of nerve growth factor receptors in PC12 cells. The data indicate that the Mr = 148 000-158 000 species affinity-labeled by 125I nerve growth factor is the native form of a component associated with kinetically different nerve growth factor receptors in PC12 cells.     

Properties of the nerve growth factor receptor of a clonal line of rat pheochromocytoma (PC12) cells.

The interaction of nerve growth factor (NGF) with its receptor on cells of the PC 12 cell line was studied. All experiments were done at 0.5 °C to minimize degradation and processes requiring membrane mobility. Under these conditions, a single class of high affinity binding sites with a dissociation constant of 2.9 × 10^?9 M was observed. The number of receptors per cell was 58000. The binding was linear with the number of cells in the assay and was not displaced by proteins other than native nerve growth factor. Trypsin treatment of the cells destroyed the specific binding. The removal of divalent cations had no effect on the binding. Culturing the cells for 2 weeks in NGF prior to assay did not change the receptor number or receptor affinity and there was a similar lack of effect of the density of the culture from which the cells were taken for assay. The present findings are compared with previous studies on the dorsal root ganglia and sympathetic ganglia neurons, and the implication for the use of PC 12 as a model for the study of NGF action are discussed.     

Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a polyclonal antiserum directed against a putative 140-kDa fibroblast cell surface fibronectin receptor (anti-gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti-gp 140 or its Fab fragments, NGF-stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS-PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120-140-kDa band, suggesting that the site of anti-gp140 action in PC12 cells is also through a fibronectin receptor.     

Nerve growth factor induces 5-HT3 recognition sites in rat pheochromocytoma (PC12) cells

In rat pheochromocytoma (PC12) cells, nerve growth factor (7S NGF) induced the expression of recognition sites that bind the specific 5-HT3 antagonist (S-) [3H]zacopride. Culturing PC12 cells for 8-12 days in the presence of 50 ng/ml NGF increased the density (Bmax) of (S-) [3H]zacopride binding sites in cell membranes (0-100,000 x g fraction) from 0 to 105 fmoles/mg protein. This binding exhibited high affinity for (S-) [3H]zacopride (Kd = 0.8 nM), was specific (greater than 95%), and was inhibited by 5-HT3 compounds with a rank of potency (quipazine greater than ICS 205-930 greater than GR38032F greater than BRL24924 approximately MDL 72222 greater than phenylbiguanide greater than or equal to serotonin greater than 2-methyl-serotonin greater than metoclopramide) which was distinct from neuroblastoma cells. Thus, NGF-differentiated PC12 cells possess a 5-HT3 receptor and should be useful to investigate its regulation and biochemical mechanism of action.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

Leumorphin has an anti-apoptotic effect by activating epidermal growth factor receptor kinase in rat pheochromocytoma PC12 cells

Endogenous opioid peptides, found in the central and peripheral nervous systems, perform neuromodulatory roles, and display a wide range of functional and pharmacological properties in vitro and in vivo. In this study, we investigated the effects of prodynorphin gene products on intracellular signaling events and cell survival in rat pheochromocytoma PC12 cells. Leumorphin, but not other prodynorphin gene products including dynorphin A, beta-neoendorphin and rimorphin (dynorphin B), increased cell viability in PC12 cells. The cytoprotective effect of leumorphin was dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, but was insensitive to both naloxone, a general antagonist of the opioid receptor, and nor-binaltorphimine, a specific antagonist of the kappa opioid receptor. Moreover, a competition-binding assay clearly revealed that leumorphin had another binding site(s) in addition to that for the kappa opioid receptor. Interestingly, leumorphin induced activation of the epidermal growth factor receptor via a Src-dependent mechanism, which was proved to be responsible for the increased survival response. Flow cytometric and microscopic analysis showed that leumorphin rescued cells from serum deprivation-induced apoptosis. Collectively, we suggest that leumorphin prevents apoptosis via epidermal growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, which occur independent of the kappa opioid receptor.     

Calcium-dependent activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells by nerve growth factor

Glycogen phosphorylase in PC12 cells exists in two forms analogous to those found in brain and muscle. The active phosphorylated form of the enzyme, phosphorylase-a, represents about 20-30% of total glycogen phosphorylase in these cells. Incubation of PC12 cells with 100 ng 7S nerve growth factor/ml increased phosphorylase-a within minutes. In contrast to nerve growth factor, insulin (6 ng/ml) and epidermal growth factor (6 ng/ml) decreased phosphorylase-a. Activation of phosphorylase-a by nerve growth factor was not accompanied by increases in cyclic AMP; however, removal of extracellular Ca2+ or incubation of cells with calcium channel blockers inhibited activation of glycogen phosphorylase by nerve growth factor.     

Insulin-like growth factors decrease catecholamine content in PC12 rat pheochromocytoma cells.

Insulin-like growth factors (IGFs) stimulate proliferation and differentiation of PC12 rat pheochromocytoma cells and modulate catecholamine release in bovine adrenal medullary cells. Dexamethasone increases catecholamine synthesis in PC12 cells. We therefore studied the effects of IGFs and dexamethasone on catecholamine content in PC12 cells. Dopamine (DA) and norepinephrine (NE) content of PC12 cells were measured after incubation for 72 h with IGFs (100 ng/ml) and/or dexamethasone (500 nM). IGF-I (100 ng/ml) and IGF-II (100 ng/ml) decreased DA and NE content to approximately 35% and approximately 25% of control, respectively. [Leu27]IGF-II, which binds to the IGF-I receptor with markedly decreased affinity, did not reduce catecholamine levels, indicating that the effect is likely to be mediated by the IGF-I receptor. Dexamethasone (500 nM) increased levels of DA and NE to 173 +/- 20% and 331 +/- 48% of controls, respectively. Coincubation with IGFs did not significantly affect the stimulation of DA by dexamethasone, but abolished the rise in NE. Levels of tyrosine hydroxylase mRNA, protein and activity were increased following incubation with dexamethasone, but were unchanged by IGFs. These results indicate that IGFs decrease catecholamine content in PC12 cells via the IGF-I receptor. Complex regulation involving multiple synthetic and/or degradative steps is implicated in this process.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on fibronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment P1, corresponding to the center of the cross, and only poorly on fragment E8, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment P1, and fibronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular massess of 180 and 135 kDa. The latter is recognized by an antiserum to integrin beta 1 subunit. The bound laminin receptor could be displaced by EDTA, but not by Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to P1. The expression of the 180-kDa alpha subunit of the laminin receptor at the cell surface was increased 10-fold after NGF treatment. The effect of NGF is specific since the amount of a 150-kDa fibronectin-binding integrin alpha subunit remained unchanged. Moreover, the increased expression of the 180/135 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment P1. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on laminin.     

Insulin-like growth factors are mitogens for rat pheochromocytoma PC 12 cells

We have addressed the issue of a mitogenic effect of insulin-like growth factors IGF-I and IGF-II on the PC 12 line of rat pheochromocytoma cells. The proliferation of PC 12 cells cultured in serum-free medium is stimulated threefold by IGF-I and IGF-II with significantly higher potency than epidermal growth factor, whereas platelet-derived growth factor, nerve growth factor, growth hormone and bombesin are inactive. Two types of IGF receptor are present in PC 12 cells and the dose-response curves suggest that the mitogenic responses to IGF's are mediated by the IGF-I receptor. These results suggest that IGF-I and IGF-II act as mitogens on pluripotent chromaffin cells in the development of the peripheral nervous system and adrenal medulla as well as in promotion of in vivo growth of neural crest-derived tumors.     

Characterization of epidermal growth factor receptor function in lysophosphatidic acid signaling in PC12 cells

Lysophosphatidic acid (LPA) is a lipid metabolite that induces the activation of mitogen-activated protein kinase (MAPK) through binding to the G protein-coupled receptor in a number of cell lines and cultures. Recent studies have revealed that LPA is able to rapidly induce the phosphorylation of MAPK through an epidermal growth factor (EGF) receptor-dependent pathway. We investigated the role of the EGF receptor in the signaling pathway initiated by LPA stimulation in nerve growth factor (NGF)-responsive PC12 cells well known to transiently retract their own neurites upon LPA stimulation. LPA-stimulated MAPK signaling was suppressed by the selective EGF receptor inhibitor and in the dominant negative mutant EGF receptor cell line. As in the EGF signaling pathway, the complex of EGF receptor with adapter proteins Shc and Sos was formed in response to LPA stimulation, suggesting there is an intracellular mechanism for transactivation. A neurite retraction assay was also performed to examine the role of the EGF receptor in PC12 cell differentiation, which related to the involvement of LPA-induced neurite retraction. These results suggest that the receptor tyrosine kinase can be activated in a ligand-independent manner through intracellular crosstalk between the signaling pathways.     

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.     

UDP-galactose:globotriaosylceramide alpha-galactosyltransferase activity in rat pheochromocytoma (PC12h) cells.

The activity of alpha-galactosyltransferase in cultured rat pheochromocytoma subcloned (PC12h) cells was examined using Gb3 as the acceptor for the galactose from UDP-galactose. The major reaction product was identified as gal alpha 1-3Gb3 based on its mobility on thin-layer chromatographic (TLC) plates and susceptibility to specific galactosidases. The enzyme activity in PC12h cells was the highest at pH 7.0 while the presence of Triton CF-54 (0.1%) and Mn2+ (5 mM) was required for its full activity. The apparent Km values for Gb3 and UDP-galactose were 57 and 17 microM, respectively. The enzyme activity in PC12h cells was compared with that in parent PC12 cells, in which gal alpha 1-3Gb3 is not expressed in an appreciable amount. In the enzyme reaction with exogenous Gb3, the enzyme activity in PC12h cells was about 1.5-fold higher than that in PC12 cells. In the absence of exogenous Gb3, this difference became even more pronounced; gal alpha 1-3Gb3 was generated from endogenous Gb3 at a much higher rate in PC12h cells than in PC12 cells. These findings suggest that the higher level of the alpha-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having gal alpha 1-3 terminal residues in the cells.     

Proteomic analysis of rat pheochromocytoma PC12 cells

PC12 cell line is well documented and widely applied as many kinds of models in neurobiological and neurochemical studies. Yet a thorough proteomic analysis has not been performed so far. Here we report the construction of a large-scale 2-D protein database for PC12 cells. The proteins extracted from PC12 cells were separated by 2-DE and identified by MALDI-TOF/TOF MS. A total of 1080 protein spots, excised from three different 2-D gels, were identified with high confidence. These proteins represent 474 different gene products, mainly binding proteins and enzymes. Three hundred and seven identified protein spots were located in the low-molecular weight region below 20 kDa. This database today represents one of the largest 2-D databases for higher eukaryotic cell proteomes and for low-molecular weight proteins. In addition, fragment ion spectra obtained by TOF/TOF confirmed that calcylin in PC12 cells was N-acetylated. The database of PC12 proteome is expected to be a powerful tool for neuroscientists.     

Selective effect of nerve growth factor on some Golgi and lysosomal enzyme activities of rat pheochromocytoma (PC12) cells

Treatment with neuronal growth factor (NGF) results in the growth of neuronal processes by PC12 cells and a concomitant 70% increase in the area of the Golgi apparatus. To define the observed morphologic changes in biochemical terms, we investigated the effect of NGF treatment on some Golgi and lysosomal enzyme activities of PC12 cells. Enzyme activities characteristic of the Golgi apparatus, lysosomes, plasma membranes, mitochondria, and endoplasmic reticulum were measured in cell homogenates, in post-mitochrondrial supernatants, and in Golgi-enriched fractions from control and from NGF-stimulated PC12 cells. Treatment of PC12 cells with NGF did not change the level of the Golgi activity of UDPGal:GlcNAc galactosyltransferase while that of CMP-sialic acid:lactosylceramide sialyltransferase was increased three- to fivefold in all fractions studied. For lysosomal enzymes, NGF treatment resulted in a two- to threefold higher level of arylsulfatase activity compared to either acid phosphatase or acid alpha-mannosidase activities. These results indicate that there is a selective increase of at least one Golgi and one lysosomal activity as a result of NGF stimulation of PC12 cells. Both of these enzymes are involved in glycolipid metabolism. It is possible that the dramatic morphologic changes observed during NGF-induced differentiation of PC12 cells are associated not only with increased synthesis in the Golgi apparatus of plasma membrane components such as gangliosides, but also with increased degradation in lysosomes of other plasma membrane components such as sulfatide.     

Calcium channels in undifferentiated PC12 rat pheochromocytoma cells

Undifferentiated rat pheochromocytoma PC12 cells were voltage clamped using the whole cell technique. After blockade of outward currents, calcium currents were elicited from -40 and -100 mV. A subpopulation of cells displayed only one current component activated at -10 mV and slowly decaying. In other cells this current coexisted with a component activated around -40 mV and decaying with a faster time constant. We conclude that undifferentiated PC12 cells can express two types of calcium channels, L (long-lasting) and N (neuronal)-type channels.     

The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.