PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division.

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.

Differential utilization of beta-tubulin isotypes in differentiating neurites.

beta-Tubulin is encoded in vertebrate genomes by a family of six to seven functional genes that produce six different polypeptide isotypes. We now document that although rat PC-12 cells express five of these isotypes, only two (classes II and III) accumulate significantly as a consequence of nerve growth factor-stimulated neurite outgrowth. In contrast to previous efforts that have failed to detect in vivo distinctions among different beta-tubulin isotypes, we demonstrate using immunoblotting with isotype-specific antibodies that three beta-tubulin polypeptides (classes I, II, and IV) are used preferentially for assembly of neurite microtubules (with approximately 70% of types I and II assembled but only approximately 50% of type III in polymer). Immunofluorescence localization shows that an additional isotype (V) is partially excluded from neurites. Distinctions in in vivo localization of the neuron-specific, class III isotype have also been directly observed using immunofluorescence and immunogold electron microscopy. The sum of these efforts documents that some in vivo functional differences between tubulin isotypes do exist.

Methionine-enkephalin and leucine-enkephalin in human sympathoadrenal system and pheochromocytoma.

To elucidate the physiological and pathophysiological significance of methionine- and leucine-enkephalin (Met-and Leu-enkephalin, respectively) in human sympathoadrenal system, the contents of these peptides in normal human sympathetic nervous system, adrenal medulla, and pheochromocytomas were determined by specific radioimmunoassays combined with reverse-phase high-performance liquid chromatography. Met-enkephalin-LI and Leu-enkephalin-LI, respectively) were detected by radioimmunoassay in adrenal glands, adrenal medulla, stellate ganglia, sympathetic trunks, and celiac ganglia, and their contents in adrenal medulla were highest. Existence of authentic Met- and Leu-enkephalin was confirmed by reverse-phase high-performance liquid chromatography. Met-enkephalin was approximately 74% of Met-enkephalin-LI, whereas Leu-enkephalin was approximately 30% of Leu-enkephalin-LI in human adrenal medulla. The ratio of Met- to Leu-enkephalin was 2.6 in human adrenal medulla, whereas it was higher in sympathetic ganglia or trunks. In four cases of pheochromocytoma marked difference in Met- and Leu-enkephalin contents was found between medullary and extramedullary tumors. The contents were about three orders higher and the Met- to Leu-enkephalin ratio was lower in medullary than in extramedullary pheochromocytomas, reflecting the tissues where the tumors arose. These results suggest the physiological roles of Met- and Leu-enkephalin in sympathetic nervous system and adrenal glands and their pathophysiological significances in pheochromocytomas.

Production of the alpha subunit of guanine nucleotide-binding protein GO by neuroendocrine tumors.

We have found that neuroendocrine tumors (including neuroblastoma, ganglioneuroma, gut carcinoid, pheochromocytoma, medullary thyroid carcinoma, insulinoma, glucagonoma, prolactinoma, carotid body tumor, and small cell lung carcinoma) produce considerable amounts (about 1000-80,000 ng/g tissue) of the alpha subunit of guanine nucleotide-binding protein, GO (GO alpha), whereas nonneuroendocrine tumors contain less than 300 ng of GO alpha/g tissue. GO alpha in the neuroendocrine tumors was present both in the soluble fraction, and cholate-extractable membrane-bound fraction of tissues. Immunoblots of membrane fractions of neuroblastoma and carcinoid tissues confirmed that the immunoreactive substance in the tumor tissues was GO alpha. Immunohistochemically, GO alpha was localized consistently in the cell membrane and occasionally in the cytoplasm of neuroendocrine tumors. GO alpha was also detected in sera of 73% patients with neuroblastoma at diagnosis, whereas serum GO alpha concentrations in control children, or patients with nonneuroendocrine tumors were lower than the detection limit of the immunoassay method employed. Serum GO alpha concentrations in patients with neuroblastoma changed with the clinical course; they fell in patients responding to treatment and increased in patients who relapsed. Since GO alpha, a specific protein in the neural and neuroendocrine cells, was found to be produced in considerable amounts by all types of neuroendocrine tumors but not in nonneuroendocrine tumors, GO alpha might be a useful biomarker for neuroendocrine tumors.

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling.

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.

Familial extra-adrenal pheochromocytoma. A new syndrome.

Pheochromocytomas in the same anatomic site, the right renal hilum, occurred in a family over three successive generations. For two patients in the latter two generations, scintigraphy with iodine 131-tagged metaiodobenzylguanidine (MIBG) showed tumors only in the region of the right renal hilum, thus indicating that they were primary lesions. At surgery, except for lymph node metastases noted microscopically in one patient, tumors were found only near the right renal hilum. The adrenal glands seemed normal on inspection, palpation, and computed tomography. In another family, a mother and son had primary pheochromocytomas arising from the urinary bladder. We suggest that primary extra-adrenal pheochromocytoma is a syndrome in which specific genetic abnormalities determine sites of tumor development.

Two forms of parathyroid hormone-like adenylate cyclase-stimulating protein derived from tumors associated with humoral hypercalcemia of malignancy.

Tumors associated with humoral hypercalcemia of malignancy (HHM) contain parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs). We previously purified a 17,000 MW hACSP from an HHM-associated breast carcinoma. This report describes the characterization of hACSPs from three additional HHM-associated tumors: two typical HHM-associated tumors (squamous carcinomas) and a third unusual tumor type (pheochromocytoma). Each tumor was extracted in acid-urea/ethanol-sodium chloride, and adenylate cyclase-stimulating activity (ACSA) was examined following reverse-phase and size-exclusion HPLC and isoelectric focusing. Each tumor contained a high molecular weight form of ACSA which co-eluted with the 17,000 molecular weight breast carcinoma hACSP in each of the three separation procedures. Each also contained a lower molecular weight form(s) of ACSA (6,000-9,000 molecular weight). Both forms were inhibited by Nle8,18Tyr34bPTH(3-34) amide. The high molecular weight form was not changed to the lower molecular weight form by a reducing agent. Some HHM-associated tumors contain two forms of hACSP, one with a molecular weight of 17,000 and another with a molecular weight of 6,000-9,000, which appears to be an amino-terminal cleavage product of the larger protein.

Localization and molecular forms of galanin in human adrenals: elevated levels in pheochromocytomas.

Galanin immunoreactivity was measured by RIA, using antibodies directed against both the non-C- and C-terminal positions of porcine galanin, in tissue extracts of normal adrenals and pheochromocytomas and also in the plasma of normal subjects and patients with pheochromocytomas. No C-terminal galanin-like immunoreactivity was detected in plasma or tissue, suggesting differences in the amino acid sequence of human compared with porcine galanin. A non-C-terminally directed antibody was, therefore, used to characterize human galanin immunoreactivity by gel permeation chromatography and reverse phase high pressure liquid chromatography and to localize it by immunocytochemistry. The galanin content of whole adrenal gland was 2.6 +/- 0.9 (+/- SEM) pmol/g (n = 5). In contrast, however, pheochromocytomas had much greater concentrations (21 +/- 2.3 pmol/g; n = 16). Gel chromatography and reverse phase high pressure liquid chromatography revealed 2 molecular forms of galanin immunoreactivity with identical elution positions in both normal adrenals and tumors. The concentration of galanin in plasma from both normal subjects and pheochromocytoma patients was below the detection limit of the assay (less than 10 pmol/liter). Using immunocytochemistry, galanin was localized to scattered cells or clusters of tumor cells in 5 of 11 pheochromocytomas and only a few chromaffin cells and cortical nerve fibers in normal adrenals.

Expression of c-fos and c-myc proto-oncogenes in human adrenal pheochromocytomas.

We examined c-fos and c-myc expressions in pheochromocytoma tissues from six patients. All samples contained c-fos and c-myc transcripts, whereas mRNA from bovine adrenal medulla, as a control, did not contain these transcripts at detectable levels. Southern blot analysis revealed no amplification and no rearrangement of c-fos and c-myc genes. We also examined the gene expression of insulin-like growth factor-II (IGF-II), a mitogen for rat pheochromocytoma cells exerted by autocrine or paracrine fashion. All samples from the pheochromocytomas contained IGF-II transcripts as well as c-fos and c-myc transcripts. The constitutive expressions of c-fos and c-myc genes may be interpreted to mean that pheochromocytoma is in a state of growth stimulation in vivo by growth factors, including IGF-II.

Neurohypophysial hormones in the adrenal medulla.

Immunoreactive oxytocin and arginine vasopressin (AVP) were measured in the adrenal medulla of both rat and man as well as in tissue from two pheochromocytomas using highly specific RIAs. In all instances, oxytocin predominated over AVP. The concentrations of oxytocin ranged from 19.9-162.7 pg/g tissue, whereas those for AVP were 9.8-102 pg/g. These values are far greater than those found in plasma. The oxytocin- and AVP-related neurophysins were also present in large quantities in human adrenal medulla and pheochromocytoma. Identity of the peptides was confirmed by demonstrating parallel immunoreactivity with standard compounds and by the high performance liquid chromatographic profiles. In experiments carried out in rats, the source of the adrenal medullary AVP and oxytocin did not appear to be the paraventricular nucleus. It is postulated that the neurohypophysial peptides may have a regulatory function in the secretion of catecholamines.

Immunoreactive corticotropin and corticotropin-releasing factor in human hypothalamus, adrenal, lung cancer, and pheochromocytoma.

Immunoreactive corticotropin-releasing factor (I-CRF) and ACTH (I-ACTH) were examined using RIA, immunoaffinity chromatography, and gel filtration chromatography in human hypothalamus, adrenal (cortex and medulla), lung cancer, and pheochromocytoma. I-CRF and I-ACTH were present in these tissues. Gel filtration of I-ACTH in the adrenal, pheochromocytoma, and lung cancer showed the presence of larger amounts of I-ACTH with large molecular weight forms in contrast to the hypothalamus. Gel filtration of I-CRF in these tissues showed the main peak eluted at the position of synthetic rat CRF. High performance liquid chromatography of this main peak showed two components which eluted in the positions of synthetic rat CRF and oxidized CRF. These elution positions were the same in all tissues and identical with those in the hypothalamus. These results suggest the presence of I-ACTH and I-CRF in these tissues and that CRF outside the brain is identical to hypothalamic CRF.

Immunohistological localization of growth hormone-releasing hormone in human tumors.

Two forms of GH-releasing factor (GHRH), which play a role in the regulation of GH secretion, have been isolated from pancreatic endocrine tumors in two patients with acromegaly. We examined formalin-fixed, paraffin-embedded human tissues from autopsies and surgical specimens for the presence of human pancreatic GHRH-40 using the avidin-biotin-peroxidase complex technique to assess the prevalence of tumors containing GHRH, to define their primary sites and cellular derivations, and to correlate clinical and pathological features. Immunopositivity was demonstrated in 4 of 24 pancreatic endocrine tumors, 1 of 5 bronchial and 2 of 15 gut carcinoids, 1 of 2 thymic carcinoids, 2 of 20 medullary carcinomas of the thyroid, 1 of 12 pheochromocytomas, and 5 of 20 small cell carcinomas of the lung. Of the GHRH-containing tumors, only 2 of the pancreatic endocrine tumors and the bronchial carcinoid were associated with acromegaly. No GHRH was found in 35 tumors derived from cells that are not known to produce peptide hormones. Immunoreactivity was not detected in the nontumorous tissues from which GHRH-containing tumors were derived. It can be concluded that GHRH may be found in a variety of tumors arising from and composed of peptide hormone-producing endocrine cells. The significance of immunoreactive GHRH detected in tumors unassociated with clinical evidence of acromegaly remains to be established.

Immunoreactive human chromogranin A in diverse polypeptide hormone producing human tumors and normal endocrine tissues.

Chromogranin A, the major soluble protein costored and coreleased by exocytosis with catecholamines from the adrenal medulla, has recently been detected in several bovine polypeptide hormone producing tissues. We therefore searched for chromogranin, by immunohistology, in human polypeptide hormone producing tumors as well as normal human endocrine tissues. The chromogranin A antigen was purified from catecholamine storage vesicles of human pheochromocytoma, to which rabbit antisera were developed, allowing immunohistologic studies by the indirect rabbit anti-peroxidase technique. Specific chromogranin staining was noted in all polypeptide hormone producing human tumors studied (pheochromocytoma chromaffin cells, n = 3; medullary thyroid carcinoma parafollicular C cells, n = 2; thyroidal C cell hyperplasia cells, n = 1; parathyroid adenoma chief cells, n = 1; pancreatic islet cell tumor islet cells, n = 1; oat cell carcinoma cell line M-103) as well as in all normal polypeptide hormone producing tissues (adrenal medulla chromaffin cells, parathyroid chief cells, thyroid parafollicular C cells, pancreatic islet cells, gut enteroendocrine cells, and anterior pituitary cells). Chromogranin may have a widespread distribution in human polypeptide hormone producing tissues, and may be a useful histologic marker for peptide producing tumors.

Immunoreactive corticotropin-releasing hormone, growth hormone-releasing hormone, somatostatin, and peptide histidine methionine are present in adrenal pheochromocytomas, but not in extra-adrenal pheochromocytoma.

CRH, GH-releasing hormone (GHRH), somatostatin (SRIH), and peptide histidine methionine (PHM) were measured by RIA in extracts of normal adrenal glands and in extracts from adrenal and extraadrenal pheochromocytomas. In normal adrenal glands, immunoreactive (IR) CRH, IR-SRIH, and IR-PHM were detectable, while IR-GHRH was undetectable. In all 11 cases of adrenal pheochromocytomas, the tumors contained 2 or more of these four IR-peptides. In particular, IR-CRH was found in 73% (n = 8) of adrenal pheochromocytomas, IR-GHRH in 91% (n = 10), IR-SRIH in 91% (n = 10), and IR-PHM in 82% (n = 9) of adrenal pheochromocytomas. There was no significant correlation among the concentration of these peptides in each tumor, i.e. the concentrations of the IR-peptides were independent of each other. In contrast to the adrenal pheochromocytomas, none of these 4 IR-peptides was detectable in 5 extraadrenal pheochromocytomas. Gel filtration of pooled extracts from adrenal pheochromocytomas showed that the major component of the IR-CRH, IR-GHRH, IR-SRIH, and IR-PHM eluted in the position of their synthetic counterparts. Our results suggest that 1) the normal adrenal gland contains IR-CRH, IR-SRIH, and IR-PHM, but not IR-GHRH; 2) all of the adrenal pheochromocytomas we examined contained a number of hypothalamic releasing or inhibiting hormones; 3) their tissue concentrations were independent of each other; and 4) all of the extraadrenal pheochromocytomas we examined contained no such IR-peptides. The presence of hypothalamic hormones in adrenal pheochromocytomas and their absence in extraadrenal pheochromocytomas may be due to the differences in the chromaffin cells of their origin. Our data may be helpful in the differential diagnosis between adrenal and extraadrenal pheochromocytomas.

Human chromogranin A. Purification and characterization from catecholamine storage vesicles of human pheochromocytoma.

Chromogranin A is the quantitatively major soluble protein in catecholamine storage vesicles of the adrenal medulla and sympathetic nerve, and has been a useful index of exocytosis during sympathoadrenal neurosecretion. To probe human catecholamine storage and release, we isolated chromogranin A from chromaffin tissue in human pheochromocytoma, and compared it to chromogranin A isolated from chromaffin tissue in bovine adrenal medulla. The preparation included catecholamine storage vesicle isolation by sucrose gradient centrifugation, removal of dopamine-beta-hydroxylase by affinity chromatography on Concanavalin A-Sepharose, and preparative polyacrylamide gel electrophoresis. Human and bovine chromogranin A displayed considerable interspecies homology. Human chromogranin A is a 68,000 dalton monomeric protein with an unusual amino acid composition (31.53 weight % glutamic acid); an acidic, microheterogeneous isoelectric point (4.57-4.68); a characteristic tryptic digest peptide map; and marked dissimilarity to dopamine-beta-hydroxylase in all properties studied. A new probe of human sympathoadrenal function is available in chromogranin A.

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells.

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.

Occurrence of glucose polymer in undifferentiated PC12 cells.

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.

Antibodies to the beta-amyloid peptide cross-react with conformational epitopes in human fibrinogen subunits from peripheral blood.

Antibodies to the Alzheimer disease (AD) beta-amyloid peptide (beta AP) were used to identify beta AP precursor fragments in blood. The antibodies detected 3 major polypeptides with apparent molecular weights (MW) of 47-64,000 in Western blots of plasma derived clot proteins, but these proteins corresponded to human A-alpha, B-beta and gamma-fibrinogen since they reacted with 2 different anti-fibrinogen antisera, and the anti-beta AP and anti-fibrinogen antibodies recognized purified fibrinogen and fibrin. These data are significant for efforts to develop immunochemical assays to diagnose and monitor the progression of AD.

Ganglionic nAChRs and high-affinity nicotinic binding sites are not equivalent.

High-affinity (Kd approximately equal to 10 nM) binding sites for nicotine and acetylcholine (ACh) have recently been identified in vertebrate brain. It has been suggested that these sites are desensitized ganglionic (C6) nicotinic acetylcholine receptors (nAChRs). We have tested the pheochromocytoma cell line PC12, which is known to contain well-expressed C6 nAChRs, to determine if these nAChRs are associated with high-affinity [3H]ACh-binding sites. We found that the high-affinity nicotinic [3H]ACh-binding site is absent in PC12 cells. We also found that the concentration of nicotine or ACh necessary to desensitize carbamylcholine-stimulated Na+ flux was at least two orders of magnitude greater than the concentrations used in binding experiments. We conclude that high-affinity nicotinic binding sites are not equivalent to C6 ganglionic receptors.