Involvement of tetrahydrobiopterin in trophic effect of erythropoietin on PC12 cells.

Tetrahydrobiopterin (BH(4)) synthesis is reported to be stimulated by nerve growth factor (NGF) in PC12 cells, suggesting involvement of BH(4) in the trophic effect of NGF. We have recently reported that erythropoietin (EPO) and BH(4) enhance survival of PC12 cells. In the present study, we investigated involvement of BH(4) in the trophic effect of EPO on PC12 cells. Cellular BH(4) content was increased by EPO (10(-10) to 10(-8) M) in a dose- and time-related manner. EPO (10(-10) to 10(-8) M) increased the viable cell number of PC12 cells. In addition to EPO, BH(4) (1, 3, and 10 microM) increased the viable cell number of PC12 cells. Administration of 0.3 mM 2,4-diamino-6-hydroxypyrimidine, an inhibitor of BH(4) synthesis, blunted EPO-induced increases in BH(4) content and the viable cell number of PC12 cells. These results taken together suggest that BH(4) is involved in the trophic effects of EPO on PC12 cells.     

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

The effects of extracellular Na+, K+ and Cl- on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na+ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60-150 mM Na+, whereas it decreased significantly at lower concentrations of Na+. Na+ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast neurite outgrowth was independent of K+ in the range 5-106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl- replaced by NO3-, SO2-4, or 2-hydroxyethanesulfonate. Thus, extracellular Na+ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca2+ concentration monitored by a fluorescent dye sensitive to Ca2+.     

Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine-coated glass slides) by fluorescence techniques (with quin-2 and fura-2). The effect of NGF on [Ca2+]i is delayed (30-40 s of lag phase), slow (t1/2 = 40 s), relatively small (+50-75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extracellular Ca2+ greater than 10 microM) through a pathway different from the voltage-gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action.     

Ca2+ channel activating function of maitotoxin, the most potent marine toxin known, in clonal rat pheochromocytoma cells

Actions of maitotoxin, the most potent marine toxin known obtained from toxic dinoflagellate, Gambier-discus toxicus, were studied using clonal rat pheochromocytoma cells (PC12), rat liver mitochondria and liposomes. Maitotoxin induced a profound release of norepinephrine and dopamine from PC12 cells and the molar ratio of norepinephrine to dopamine was almost the same as that stored in the cells. This releasing action was apparent at a concentration of 5 X 10(-10) g/ml or more, the releasing rate increased with an increase in the concentration of applied maitotoxin and attained maximum at about 10(-6) g/ml. The [3H]norepinephrine release induced by maitotoxin was abolished in the absence of external Ca2+ and increased with increasing concentration of external Ca2+ up to 10 mM. The release gradually decreased as the external Na+ concentrations were reduced from 130 to 20 mM, but maitotoxin is still able to induce a profound release in the absence of external Na+. The releasing action of maitotoxin was markedly suppressed by various Ca2+ channel blockers, such as Mn2+, verapamil, and nicardipine, and by a local anesthetic, tetracaine. The inhibitory actions of Ca2+ channel blockers were antagonized by external Ca2+ and became less obvious in the higher Ca2+ concentration range. Maitotoxin did not exhibit any ionophoretic activities on rat mitochondrial and liposomal membranes. These results suggest that maitotoxin has the ability to activate voltage-dependent Ca2+ channels of PC12 cells.     

Induction of immediate early genes by Ca2+ influx requires cAMP-dependent protein kinase in PC12 cells

Agents that activate cAMP-dependent protein kinase (PKA) as well as agents that increase intracellular calcium induce the expression of certain immediate early genes (IEGs). Recently, it has been demonstrated that the same cis-acting element in the 5' region of the c-fos gene has the ability to mediate both cAMP- and calcium-induced c-fos expression in PC12 cells (Sheng, M., McFadden, G., and Greenberg, M. (1990) Neuron 4, 571-582). Here we demonstrate that both cAMP- and calcium-mediated induction of c-fos and egr1 are dependent on PKA activity. Addition of either depolarizing concentrations of KCl or the calcium ionophore, ionomycin, to PC12 cells increased the expression of both c-fos and egr1, but these inductions were dramatically reduced in three PKA-deficient cell lines, 123.7, AB.11, and A126-1B2. Furthermore, pretreatment of PC12 cells with 20 microM H89, a specific inhibitor of PKA, inhibited forskolin, dibutyryl cAMP, and KCl-induced c-fos and egr1 induction, while having no effect on NGF induction. Likewise, in the PKA-deficient cells, NGF or an activator of protein kinase C induced c-fos and egr1 normally. To determine if PKA deficiency modifies the ability of Ca2+ to activate calcium-dependent kinases, autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in response to Ca2+ influx was determined. In parental PC12 cells, PC12 cells pretreated with H89, and PKA-deficient cell lines, CaM kinase was activated equivalently in response to KCl depolarization. These results suggest that PKA is not required for Ca(2+)-induced increase in CaM kinase activity and that the induction of IEGs in response to Ca2+ influx is PKA-dependent. Thus, the requirement for PKA resides at a point distal to the activation of calmodulin-dependent processes.     

M6a acts as a nerve growth factor-gated Ca(2+) channel in neuronal differentiation

To elucidate the function of M6a, which is a neuron-specific membrane glycoprotein of the brain and possesses putative phosphorylation sites for protein kinase C (PKC), we established rat M6a cDNA expression vector-transfected PC12 cells. These transfectants exhibited high susceptibilities to nerve growth factor (NGF) for neuronal differentiation. Interestingly, we found that Ca(2+) influx in these transfectants was significantly augmented by the treatment of NGF, but not epidermal growth factor (EGF), which stimulates PC12 cell growth. NGF-dependent augmentation of Ca(2+) influx was detected within 3h and severely inhibited by EGTA- and PKC-specific inhibitors. Anti-M6 antibody suppressed both NGF-triggered Ca(2+) influx and neuronal differentiation. These results support the idea that M6a implicates in neuronal differentiation as a novel Ca(2+) channel gated selectively by phosphorylation with PKC in the downstream of NGF signaling pathway.     

Multiple acute effects on the membrane potential of PC12 cells produced by nerve growth factor (NGF)

We studied whether nerve growth factor (NGF) can affect the membrane potential and conductance of PC12 cells. We demonstrate that NGF depolarizes the membrane of PC12 cells within a minute and by using transfected NIH 3T3-Trk and -p75 cells we show that both the high affinity NGF receptor p140(trk) and the low affinity NGF receptor or p75(NGF) may be involved in the depolarization. Tyrosine kinase inhibitor, K252a, partially inhibited the depolarization, but two agents affecting intracellular calcium movements, Xestospongin C (XeC) and thapsigargin, did not. The early depolarization was eliminated in Na+ free solutions and under this condition, a 'prolonged' (> 2 min) hyperpolarization was observed in PC12 cells in response to NGF. This hyperpolarization was also induced in PC12 cells by epidermal growth factor (EGF). Voltage clamp experiments showed that NGF produced a late (> 2 min) increase in membrane conductance. The Ca2+-dependent BK-type channel blocker, iberiotoxin, and the general Ca2+-dependent K+ channel blocker, TEA, attenuated or eliminated the hyperpolarization produced by NGF in sodium free media. Under pretreatment with the non-selective cation channel blockers La3+ and Gd3+, NGF hyperpolarized the membrane of PC12 cells. These results suggest that three different currents are implicated in rapid NGF-induced membrane voltage changes, namely an acutely activated Na+ current, Ca2+-dependent potassium currents and non-selective cation currents.     

Depolarization-Induced Increase in Surface Binding and Internalization of 125I-Nerve Growth Factor by PC 12 Pheochromocytoma Cells

The binding and internalization of 125I-nerve growth factor (NGF) by PC12 pheochromocytoma cells was studied as a function of extracellular potassium concentration. Both surface-bound and internalized fractions of 125I-NGF associated with the cells under depolarizing conditions (50 mM K+) increased to 144 +/- 28% (average +/- SEM, six different cell preparations) and to 176 +/- 12% (n = 6), respectively, of those observed at 6.0 mM K+. Scatchard-type analysis of the data indicates increased sites for the binding and internalization of iodinated NGF by the cells. Similar enhancement was observed for cells treated with NGF as well. This voltage-dependent phenomenon was reversible, and also observed in the presence of veratridine. Moreover, withdrawal of extracellular Ca2+ abolished high K+-induced modulation of 125I-NGF binding and internalization, indicating that this effect may be mediated by Ca2+.     

Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation.

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.     

S100B increases proliferation in PC12 neuronal cells and reduces their responsiveness to nerve growth factor via Akt activation

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner.