thioguanine-anhydrous and Pheochromocytoma

The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 microM 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells.

Topics: Adrenal Gland Neoplasms; Animals; Carnitine O-Acetyltransferase; Choline O-Acetyltransferase; Nerve Growth Factors; Pheochromocytoma; Protein Kinase C; Protein Kinase Inhibitors; Rats; Thioguanine; Tumor Cells, Cultured

Purine analogues were used to probe the mechanism by which nerve growth factor (NGF) and other agents regulate cellular ornithine decarboxylase (ODC) activity. Exposure of cultured rat pheochromocytoma PC12 cells to NGF causes a 10-50-fold induction of ODC activity within 4-6 h. We recently found that purine analogues block this induction as well as other, but not all, actions of NGF and have provided evidence that the inhibitory actions of the analogues may be due in part to the suppression of an NGF-activated protein kinase activity (Volonté, C., Rukenstein, A., Loeb, D. M., and Greene, L. A. (1989) J. Cell Biol. 109, 2395-2403). The present results show that the purine analogues also suppress the induction of ODC mRNA. One of the analogues used was 6-thioguanine (6-TG). Although 6-TG was effective when applied simultaneously with NGF, if NGF was administered for as little as 1-3 min before 6-TG, ODC induction was unimpaired. This suggests that 6-TG blocks an early step in the NGF mechanism, and that once this step is triggered, the ODC induction pathway is no longer sensitive to this analogue. In contrast, another purine analogue, 2-aminopurine (2-AP), effectively inhibited ODC induction even if applied only during the last hour of a 5-h exposure to NGF. It is hypothesized that this increased period of sensitivity to 2-AP may be due to its broader range (as compared to 6-TG) as an inhibitor of protein kinase activities. Epidermal growth factor (EGF) and cAMP derivatives also induce ODC activity in PC12 cells, and these effects were suppressed by 6-TG and 2-AP at concentrations similar to those that affect responses to NGF. However, short term (less than 30 min) pretreatment with EGF or a cAMP derivative did not protect induction of ODC activity by these agents from inhibition by 6-TG. This suggests that there are both convergent and divergent elements in the mechanistic pathways used by NGF, cAMP analogues, and EGF to induce ODC.

Topics: 2-Aminopurine; Adrenal Gland Neoplasms; Animals; Cyclic AMP; Enzyme Induction; Epidermal Growth Factor; Kinetics; Nerve Growth Factors; Ornithine Decarboxylase; Pheochromocytoma; Rats; RNA, Messenger; Thioguanine; Thionucleotides; Tumor Cells, Cultured

Purine analogues were used in this study to dissect specific steps in the mechanism of action of nerve growth factor (NGF). Protein kinase N (PKN) is an NGF-activated serine protein kinase that is active in the presence of Mn++. The activity of PKN was inhibited in vitro by purine analogues, the most effective of which was 6-thioguanine (apparent Ki = 6 microM). Several different criteria indicated that 6-thioguanine is not a general inhibitor of protein kinases and that it is relatively specific for PKN. For instance, it did not affect protein kinases A or C and was without effect on the overall level and pattern of protein phosphorylation by either intact or broken PC12 cells. Since purine analogues rapidly and effectively enter cells, they were also assessed for their actions on both transcription-dependent and -independent responses of PC12 cells to NGF. NGF-promoted neurite regeneration was reversibly suppressed by the analogues and at concentrations very similar to those that inhibit PKN. Comparable concentrations of the analogues also blocked NGF-stimulated induction of ornithine decarboxylase activity. In contrast to its inhibition of neurite regeneration and ornithine decarboxylase induction, 6-thioguanine did not suppress NGF-dependent induction of c-fos mRNA expression. Thus, purine analogues such as 6-thioguanine appear capable of differentially suppressing some, but not other actions of NGF. These findings suggest the presence of multiple pathways in the NGF mechanism and that these can be dissected with purine analogues. Moreover, these data are compatible with a role for protein kinase N in certain of these pathways.

Topics: 2-Aminopurine; Adenine; Adrenal Gland Neoplasms; Animals; Axons; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Kinetics; Nerve Growth Factors; Nerve Regeneration; Ornithine Decarboxylase; Pheochromocytoma; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Thioguanine