Geneva Dickens

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.

The neurite-promoting effect of fibroblast growth factor on PC12 cells

Treatment of PC12 cells with fibroblast growth factor(s) from either brain or pituitary caused neurite outgrowth comparable to that produced by nerve growth factor. The neurite outgrowth was preceded by a substantial rise in the activity of ornithine decarboxylase.

Nerve growth factor (NGF)-induced calcium influx and intracellular calcium mobilization in 3T3 cells expressing NGF receptors

The neurotrophins have been implicated in the acute regulation of synaptic plasticity. Neurotrophin-stimulated presynaptic calcium uptake appears to play a key role in this process. To understand the mechanism of neurotrophin-stimulated calcium uptake, the regulation of calcium uptake and intracellular mobilization by nerve growth factor (NGF) was investigated using NIH 3T3 cells stably transfected with either the high affinity NGF receptor p140 trk (3T3-Trk) or the low affinity NGF receptor p75NGFR(3T3-p75). In 3T3-Trk cells, NGF increased both calcium uptake and intracellular calcium mobilization. In 3T3-p75 cells, NGF increased calcium uptake but not intracellular calcium mobilization. K-252a alone increased intracellular calcium in 3T3-Trk cells but not in 3T3-p75 cells. Nifedipine, an inhibitor of calcium uptake throughl-type calcium channels, inhibited the action of NGF on both 3T3-Trk cells and 3T3-p75 cells, indicating that both p140 trk and p75NGFR receptors are linked to nifedipine-sensitive l-type calcium channels. These studies show that either NGF receptor will support increases in intracellular calcium but that p140 trk does so by increasing both uptake and mobilization, whereas p75NGFR does so by increasing uptake only.

Both p140trk and p75NGFR Nerve Growth Factor Receptors Mediate Nerve Growth Factor-stimulated Calcium Uptake

Human p140trk and p75NGFR were transfected separately into 3T3 cells. Nerve growth factor stimulates calcium uptake into both transfectants but not into untransfected 3T3 cells. p140trk cells were stimulated maximally by 25 ng/ml; 100 ng/ml was submaximal for p75NGFR cells. K-252a inhibits the effect of NGF on p140trk cells but not on p75NGFR cells; brain-derived neurotrophic factor stimulates calcium uptake in p75NGFR cells but not in p140trk cells. The data suggest that both nerve growth factor receptors could be involved in the nerve growth factor-mediated actions of calcium on its target cells: neuronal survival, neuronal protection, and synaptic plasticity.

Expression of human p140trk receptors in p140trk‐deficient, PC12/endothelial cells results in nerve growth factor‐induced signal transduction and DNA synthesis

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal‐mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full‐length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN‐trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN‐trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF‐dissociation kinetics, are resistant to trypsin or Triton X‐100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose‐ (0.01‐10 ng/ml) and time‐ (5‐120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200‐1,000 nM K‐252a. NGF‐induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF‐stimulated signalling pathways found in other cells are activated in PC12EN‐trk cells. Staurosporine (5‐50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN‐trk cells. Rather, NGF treatment in low‐serum medium stimulated a 1.3‐ and 2.3‐fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN‐trk1 and PC12EN‐trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates. J. Cell. Biochem. 66:229‐244.

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Abstract: Soluble extracts from nerve growth factor (NGF)‐stimulated PC 12 cells prepared by alkaline lysis show a two‐to 10‐fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half‐maximal incorporation of 32P from [32P]ATP into S6 occurred after 4–7 min of NGF treatment. The partially purified NGF‐sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)‐dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF‐treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP‐dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5′‐N‐ethylcarboxamideadenosine also increases the subsequent cell‐free phosphorylation of S6. This observation suggests that cAMP‐dependent protein kinase may be involved in the phosphorylation of S6 kinase.

The Receptor-Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

Abstract: The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine β‐hydroxylation is not increased. Preincubation experiments suggest that the carbachol‐induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.

Involvement of protein kinase C in nerve growth factor- and K-252a-stimulated calcium uptake into PC12 cells

Both nerve growth factor (NGF) and K‐252a stimulate the uptake of calcium into PC12 cells. Stimulation by either is prevented by pretreatment of the cells with the tumor promoter phorbol 12‐myristate 13‐acetate (PMA), suggesting an involvement of protein kinase C in the stimulation. The effect of PMA is specific in that the calcium uptake stimulated by either the L‐type channel agonist BAY K 8644 or by ATP is not altered in PMA‐pretreated cells. An involvement of kinase C is also suggested by the inhibition of NGF‐ or K‐252a‐stimulated calcium uptake by the kinase C inhibitors staurosporine and calphostin C. Inhibition by the isoform‐specific agents GO 6976 and thymeleatoxin implicates one of the classic calcium‐sensitive isoforms of kinase C. The close similarity in the profiles of inhibition of NGF‐stimulated and K‐252a‐stimulated calcium uptake by the various effectors suggests that NGF and K‐252a act on calcium uptake through some of the same signaling elements. J. Neurosci. Res. 47:271–276, 1997.

The Effects of Nerve Growth Factor on Poly amine Metabolism in PC12 Cells

Abstract: Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S‐adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S‐adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor‐induced neurite outgrowth in the cells.