phosphorus-radioisotopes and Pheochromocytoma

In 1981, I made the strong statement that "The therapeutic approach of internally administered radiopharmaceuticals offers the potential to outmode the present approaches of conventional radiation therapy and chemotherapy." The present article updates and further supports this statement with new data, especially with the use of [131I]MIBG in several cancers and proposes a plan for application of nonsealed source radionuclide therapy to most solid tumors.

Topics: 3-Iodobenzylguanidine; Adrenal Gland Neoplasms; Adult; Antibodies, Neoplasm; Brachytherapy; Child; Child, Preschool; Humans; Iodine Radioisotopes; Iodobenzenes; Middle Aged; Neoplasms; Neuroblastoma; Pheochromocytoma; Phosphorus Radioisotopes; Radioisotopes; Radiotherapy Dosage; Risk

Two major isoforms of angiotensin II receptors, AT1 and AT2, have been defined on the basis of their ligand selectivity. While AT1 is known to mediate typical biological actions of angiotensin II as a cardiovascular regulator, the biological function of AT2 has not yet been established. In the present study using a rat pheochromocytoma cell line, which expresses AT2 exclusively, we found that angiotensin II inhibits phosphotyrosine phosphatase activity in vivo as measured by the inhibition of hydrolysis of [32P]-phosphate from the 32P-labeled synthetic peptide substrate, Raytide. This phosphotyrosine phosphatase inhibition was completely reversed by pertussis toxin, which indicates a G-protein coupled mechanism. In SDS-polyacrylamide gel electrophoresis we found that the phosphotyrosine group of an 85 kDa protein was a substrate mainly preserved, presumably as a consequence of the plausible intracellular phosphotyrosine phosphatase inhibition by angiotensin II.

Topics: Adrenal Gland Neoplasms; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Cell Membrane; Chromatography, Affinity; Guanosine Diphosphate; Imidazoles; Losartan; Oligopeptides; PC12 Cells; Pertussis Toxin; Pheochromocytoma; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphotyrosine; Protein Tyrosine Phosphatases; Pyridines; Rats; Receptors, Angiotensin; Tetrazoles; Thionucleotides; Tyrosine; Virulence Factors, Bordetella

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.

Topics: Adrenal Gland Neoplasms; Animals; Bradykinin; Cattle; Choline; Chromaffin System; Diacylglycerol Kinase; Ion Channels; Pheochromocytoma; Phosphatidic Acids; Phospholipase D; Phosphorus Radioisotopes; Phosphotransferases; Rats; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured; Type C Phospholipases

We examined the short-term regulation of the phosphorylation of the mid-sized neurofilament subunit (NF-M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of [32P]Pi into NF-M and (b) the rate of conversion of NF-M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF-M, because NGF alone did not increase NF-M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF-M. Further, an increase in calcium/phospholipid-dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF-M. This PKC activity was determined to be derived from the PKC alpha and PKC beta isozymes. Finally, when PC12 cells were rendered PKC-deficient by treatment with 1 muM TPA for 24 h, NGF maintained the ability to induce an increase in NF-M phosphorylation, though not to the level attained in cells which were not PKC-deficient. These data suggest that NGF with or without TPA stimulates NF-M phosphorylation as a result of a complex series of events which include PKC-independent and PKC-dependent pathways.

Topics: Adrenal Gland Neoplasms; Animals; Cells, Cultured; Down-Regulation; Intermediate Filaments; Isoenzymes; Nerve Growth Factors; Pheochromocytoma; Phorbol Esters; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Rats; Sympathetic Nervous System; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

There are five lactate dehydrogenase (LDH) isoenzymes, composed of various combinations of two types of subunits. LDH-5, which contains only the LDH A subunit, is known to be present in both the cytoplasm and the nucleus, to act as a single-stranded DNA-binding protein possibly functioning in transcription and/or replication, and to undergo phosphorylation of tyrosine 238 in approximately 1% of the enzyme after cell transformation by certain tumor viruses. We have characterized LDH from wild-type PC12 pheochromocytoma cells and from a PC12 variant (MPT1) that exhibits altered lactate metabolism and altered expression of multiple genes. Wild-type and MPT1 cells contain different proportions of LDH isoenzymes, with LDH-5 being more predominant in wild-type cells than in the variant. A small fraction of LDH from PC12 cells contains phosphotyrosine. Approximately 99% of the total LDH activity is located in the cytoplasm, but all of the phosphotyrosine-containing LDH is located in the nucleus. Furthermore, essentially all of the nuclear LDH contains phosphotyrosine. These results suggest that tyrosine phosphorylation can affect its role in the nucleus.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Cell Nucleus; Chromatography, Affinity; Cytoplasm; DNA-Binding Proteins; Isoenzymes; L-Lactate Dehydrogenase; Pheochromocytoma; Phosphates; Phosphorus Radioisotopes; Phosphotyrosine; Rats; Tyrosine; Vanadates

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Inositol Phosphates; Kinetics; Pheochromocytoma; Phosphates; Phosphatidylinositols; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rats; Tritium

As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339], tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated at an identical site (site A) by cyclic AMP-dependent protein kinase, the calmodulin-dependent multiprotein kinase and protein kinase C, while the calmodulin-dependent multiprotein kinase also phosphorylates another unique site (site C). Preparations of tyrosine hydroxylase purified from this source are also contaminated with traces of a fourth protein kinase which phosphorylates another unique site (site E). We have isolated tryptic peptides containing each of these sites and determined their amino acid sequences. By comparison of these data with the known cDNA sequence for rat tyrosine hydroxylase, we have been able to identify these sites as Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also phosphorylated a secondary site which was identified as ser-153. All of these phosphorylation sites are in the amino-terminal region, where there is no significant homology with the closely related enzyme, phenylalanine hydroxylase. Our data also establish that the initiator methionine is removed by post-translational processing to leave pro-2 as the amino-terminus of the mature protein. The significance of these results for the mechanism of action of extracellular signals on catecholamine biosynthesis is discussed.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Peptide Fragments; Pheochromocytoma; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Rats; Tyrosine 3-Monooxygenase

Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Methionine; Molecular Weight; Nerve Growth Factors; Pheochromocytoma; Phosphates; Phosphorus Radioisotopes; Rats; Sulfur Radioisotopes