dynorphins and Pheochromocytoma

Most adrenal chromaffin cells synthesize opioids derived from proenkephalin but not from prodynorphin. However, human pheochromocytomas and the PC12 rat pheochromocytoma cell line synthesize dynorphins. The aim of this study was to confirm the presence of the authentic prodynorphin transcript and its dynorphin product in PC12 cells. We have found that the sequence of a 458 bp cDNA fragment derived from RT-PCR amplification of total PC12 RNA was in complete accordance with the published sequence of the equivalent region of the prodynorphin gene. It encodes the potent endogenous kappa opioid agonists alpha-neo-endorphin, dynorphin A and dynorphin B. Furthermore, immunoaffinity-purified PC12 cell extracts were subjected to RP-HPLC. Most of its IR-dynorphin eluted on a peak exhibiting the retention time of similarly treated rat anterior pituitary. The expression of the prodynorphin gene in pheochromocytomas can be explained as either the result of (a) the process of dedifferentiation of chromaffin cells to pheochromocytoma which may thus cause the expression of a previously unexpressed prodynorphin or that (b) those pheochromocytomas expressing the prodynorphin gene derive from the few, centrally located chromaffin cells, which express this gene even under normal conditions.

Topics: Adrenal Medulla; Animals; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalins; Gene Expression; PC12 Cells; Pheochromocytoma; Pituitary Gland; Polymerase Chain Reaction; Protein Precursors; Rats; Sequence Analysis

Normal adrenal chromaffin cells produce delta opioid peptides while at the same time they have mainly kappa opioid receptors. This paper describes our date regarding the expression of the prodynorphin gene, the precursor of a family of endogenous kappa opioid ligands, in the PC12 rat pheochromocytoma cell line, and the effects of synthetic kappa opioid agonists and of Naloxone on various aspects of PC12 cell function including their secretion of catecholamines, proliferation and differentiation. This is the first part of a series of projects aimed at studying, (a) the conditions under which the prodynorphin gene is expressed in normal adrenomedullary cells, and (b) the physiological role of the endogenous kappa opioids in the physiology of the adrenal medulla.

Topics: Adrenal Medulla; Animals; Catecholamines; Cell Differentiation; Cell Division; Dynorphins; Gene Expression; Narcotics; PC12 Cells; Pheochromocytoma; Rats; Receptors, Opioid, kappa

The prodynorphin-derived opioids, dynorphin (DYN) and alpha-neoendorphin (alpha NE) were studied in 24 human phaeochromocytomas and related tumours. Nineteen tumours, extracted in HCl (0.1 mol/l), contained concentrations of immunoreactive DYN (ir-DYN) ranging from less than 0.5 to 794 pmol/g wet weight. None of the extracts in HCl contained ir-alpha NE (all less than 2.4 pmol/g). Sephadex G-50 gel filtration chromatography of ir-DYN in HCl (0.1 mol/l) extracts of six tumours revealed three small peaks of ir-DYN of higher molecular size (approximately 12,000, 6000 and 3000 daltons), a minor peak of ir-DYN eluting just after DYN(1-17), and a broad major peak, consisting of at least three components, which was significantly retarded and eluted after the salt volume of the column. High-pressure liquid chromatography (HPLC) of these extracts revealed multiple peaks of ir-DYN, most of which did not coelute with any synthetic DYN peptides. On both gel filtration chromatography and HPLC, one of the minor peaks coeluted with DYN(1-32). None of the peaks of ir-DYN coeluted with DYN(1-17) which had been acetylated using acetic anhydride. Extracts of the same tumours in acetic acid (0.1 mol/l) yielded similar values for ir-DYN content, but parallelism in the assay was improved. Sephadex G-50 chromatography revealed a different pattern of ir-DYN with a major peak coeluting with DYN(1-17) and, in two tumours, a minor peak coeluting with DYN(1-8). Studies with HPLC revealed, however, that substantial degradation of synthetic DYN occurred during extraction in acetic acid (0.1 mol/l) in spite of the precautions taken. Phaeochromocytomas frequently contain ir-DYN in concentrations which may approach that of the mammalian pituitary. These tumours did not, however, contain ir-alpha NE and, with the possible exception of a small amount of DYN(1-32), the ir-DYN present did not correspond with any known sequences. Thus, whilst prodynorphin is expressed in phaeochromocytomas, it does not seem to be processed to the usual end-products, and post-translational modifications therefore seem likely. Enzymatic degradation of DYN may occur during extraction in acetic acid (0.1 mol/l), and this medium should, therefore, be avoided in studies of such labile peptides.

Topics: Adrenal Gland Neoplasms; Chromatography, Gel; Chromatography, High Pressure Liquid; Dynorphins; Humans; Pheochromocytoma; Radioimmunoassay

We demonstrated the presence and the secretion in vivo and in vitro of immunoreactive preproenkephalin B-derived opioid peptides (alpha-neoendorphin, dynorphin and leumorphin) in human phaeochromocytomas. In seventeen human phaeochromocytomas and two human adrenal medullas, the tissue contents of immunoreactive preproenkephalin B-derived opioid peptides (alpha-neoendorphin, dynorphin and leumorphin) and leu-enkephalin were studied by specific RIAs. Compared with a remarkable wide distribution in amounts of immunoreactive leu-enkephalin (1063 +/- 437 pg/mg, mean +/- SE), small amounts of immunoreactive alpha-neoendorphin (22.6 +/- 6.4 pg/mg) and dynorphin (8.5 +/- 1.2 pg/mg) were detected in all seventeen human phaeochromocytomas and the two human adrenal medullas. Leumorphin-like immunoreactivity was detected in only four tumours. Gel chromatographic studies revealed the presence of preproenkephalin B-derived peptides and their high molecular forms. A significant positive correlation between the tumour tissue contents of immunoreactive alpha-neoendorphin and of dynorphin was observed. Nicotine (10(-5), 10(-4) mol/l) significantly stimulated the secretion of immunoreactive alpha-neoendorphin and dynorphin as well as leu-enkephalin and catecholamines from cultured human phaeochromocytoma cells. Administration of 1 mg of glucagon to a patient with medullary phaeochromocytoma induced a rapid increase in the plasma concentration of immunoreactive alpha-neoendorphin with a concomitant increase in plasma catecholamines. These results indicate the presence of preproenkephalin B-derived opioid peptides in human phaeochromocytomas and human adrenal medullas and their secretion in human phaeochromocytomas.

Topics: Adolescent; Adrenal Gland Neoplasms; Adult; Cells, Cultured; Dynorphins; Endorphins; Enkephalins; Epinephrine; Female; Glucagon; Humans; Male; Middle Aged; Nicotine; Norepinephrine; Pheochromocytoma; Protein Precursors; Radioimmunoassay

Multiple forms of immunoreactive dynorphin (I-Dy) in human pituitary and pheochromocytoma were examined utilizing gel filtration and high performance liquid chromatography (HPLC). Gel filtration of I-Dy from these tissues revealed the major component in the position of Dy(1-17) and other minor components with large molecular weight forms. HPLC profile of this major component from gel filtration showed a large peak corresponding to the position of Dy(1-17) and small peaks corresponding to the positions of Dy (1-13), (1-12) and other unknown peptides. These results strongly suggest the presence of Dy(1-17) as the major component, and Dy (1-13), (1-12) or other unknown peptides as the minor components in these human tissues.

Topics: Adrenal Gland Neoplasms; Chromatography, Gel; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Humans; Narcotics; Peptide Fragments; Pheochromocytoma; Pituitary Gland

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in two extramedullary pheochromocytomas and a medullary pheochromocytoma. The contents of dynorphin-LI of two extramedullary and a medullary tumors were 9.2, 2.2, and 34.0 pmole/g, respectively. Gel chromatographic studies of tumor extracts on Sephadex G-50 revealed a peak of dynorphin-LI eluting at the same position as dynorphin-LI contaminated in porcine ACTH extract (Sigma) and another portion of dynorphin-LI eluting in a range of molecular weights larger than that of dynorphin-LI in porcine ACTH extract. These results indicate the presence of dynorphin and possibly its precursor peptide(s) in pheochromocytoma.

Topics: Adrenal Gland Neoplasms; Adrenal Medulla; Adrenocorticotropic Hormone; Chromatography, Gel; Dynorphins; Endorphins; Humans; Molecular Weight; Pheochromocytoma