Adrenal Medullary Pheochromocytoma Research Articles

The development of the multi-physical model for cell-laden nerve scaffolds and its bioprinting by stereolithography

3D bioprinting is a novel manufacturing technology that can directly stack biocompatible materials mixed with cells layer by layer to form parts, enabling complex shapes with high resolution, and has become a powerful tool for fabricating nerve scaffolds. During the in vitro culture of scaffolds, the growth of nerve cells is affected by factors such as nutrient solution flow, oxygen concentration and scaffold structure. In this paper, a multi-physical model that combines the oxygen diffusion effect, and substrate consumption kinetics in the pc-12 (the rat adrenal medullary pheochromocytoma) cell-laden nerve scaffold was established to simulate and predict the distribution of oxygen concentration and cell growth. The scaffold was fabricated by the stereolithography (SLA) method, and the cell distribution was observed by fluorescence staining experiments to verify the model. It is approved that the model could intuitively predict cell growth. And in the future, the environmental and structural parameters of the scaffold can be optimized based on the model to achieve high density and uniform distribution of cells.

Effects of melatonin on PBDE-47-induced abnormal autophagy and apoptosis in PC12 cells

To explore the effect of melatonin (MT) on 2, 2', 4, 4'-tetrabromodiphenylether (PBDE-47)-induced abnormal autophagy and apoptosis in rat adrenal medullary pheochromocytoma PC12 cells. PC12 cells were pretreated with a concentration gradient (12.5, 25, 50, 100, and 200 μmol/L) of melatonin for 2 h before exposure to 20 μmol/L PBDE-47 for 24 h to determine the optimal concentration of melatonin for cell treatment. In subsequent experiments, PC12 cells were treated with 0.5‰ DMSO (control group), 20 μmol/L PBDE-47, 25 μmol/L melatonin, or both PBDE-47 and melatonin. Immunofluorescence assay was used to detect the positive staining of microtubule associated protein 1 light chain 3 (LC3; a marker protein of autophagy); Western blotting was performed to determine the expression levels of the key autophagic proteins including autophagy-related protein 7 (ATG7), LC3-Ⅱ and autophagy substrate p62, and the key apoptotic proteins including active cysteine-containing aspartate specific protease-3 (active caspase-3) and cleaved poly(ADP ribose) polymerase (cleaved PARP). PBDE-47 treatment significantly reduced the viability of PC12 cells (P=0.001), but pretreatment with 25 μmol/L melatonin maintained a cell viability over 80% following exposure to PBDE-47 (P=0.023). PBDE-47-treated PC12 cells showed obviously enhanced immunofluorescent staining of LC3 protein, a significantly decreased expression of ATG7 and increased expression levels of p62, LC3-Ⅱ, active caspase-3 and cleaved PARP (P < 0.001). The cells treated with both PBDE-47 and melatonin showed obviously reduced staining of LC3 protein with a signficantly increased expression level of ATG7 (P=0.034) and decreased expressions of p62 (P=0.048), LC3-Ⅱ (P=0.018), active caspase-3 (P < 0.001) and cleaved PARP (P=0.032). PBDE-47 exposure impairs autophagy to cause autophagosome accumulation and promote apoptosis of PC12 cells. Melatonin can improve PBDE-47-induced abnormal autophagy and apoptosis and thus promote the survival of PC12 cells.

Mass Spectrometry-Based Neuropeptidomics of Secretory Vesicles from Human Adrenal Medullary Pheochromocytoma Reveals Novel Peptide Products of Prohormone Processing

Neuropeptides are required for cell-cell communication in the regulation of physiological and pathological processes. While selected neuropeptides of known biological activities have been studied, global analyses of the endogenous profile of human peptide products derived from prohormones by proteolytic processing in vivo are largely unknown. Therefore, this study utilized the global, unbiased approach of mass spectrometry-based neuropeptidomics to define peptide profiles in secretory vesicles, isolated from human adrenal medullary pheochromocytoma of the sympathetic nervous system. The low molecular weight pool of secretory vesicle peptides was subjected to nano-LC-MS/MS with ion trap and QTOF mass spectrometry analyzed by different database search tools (InsPecT and Spectrum Mill). Peptides were generated by processing of prohormones at dibasic cleavage sites as well as at nonbasic residues. Significantly, peptide profiling provided novel insight into newly identified peptide products derived from proenkephalin, pro-NPY, proSAAS, CgA, CgB, and SCG2 prohormones. Previously unidentified intervening peptide domains of prohormones were observed, thus providing new knowledge of human neuropeptidomes generated from precursors. The global peptidomic approach of this study demonstrates the complexity of diverse neuropeptides present in human secretory vesicles for cell-cell communication.

177 Differentiation-Dependant Sensitivity Of Neuronal Precursor Cells Towards Oxidative Stress

Background: Apoptosis is an endogenous cell suicide mechanism triggered in response to biological factors and genotoxic stimuli often resulting from oxidative stress. Neuronal apoptosis especially in the developing brain may cause long-term brain dysfunction. Cognitve deficits in the later childhood are frequent in preterm infants. Preterm infants are exposed to high oxygen due to their premature lungs. At the same time their nutritional regimens are deficient in antioxidant precursors. In cell cultures a relation between oxidative stress and cell death could be found. This study tested the hypothesis that differentiated rat adrenal medullary pheochromocytoma PC12 cells are less sensitive to hyperoxia than undifferentiated cells.Methods: PC12 cells differentiated with nerve growth factor protein (NGF) and undifferentiated cells were exposed to an atmosphere of 80% oxygen and treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. Control cells did not receive BSO. Cell viability was tested by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide). Apoptosis was measured by flow cytometry (annexin/ propidiumjodid, caspase-3-activity).Results: Undifferentiated cells treated with BSO exposed to hyperoxia showed an increase in apoptosis (cytometry) and a significant decrease in viability (BSO+normoxia 81±5,1%, hyperoxia 95±3,1%, BSO+hyperoxia 22±3,0%, means ± S.E.M. for 4– 6 experiments, p< 0,05, cell viability assay). Differentiated cells showed reduced sensibility towards BSO and hyperoxia (BSO+normoxia 101±3,3%, hyperoxia 97±1,9%, BSO+hyperoxia 87,6±2,5%, cell viability assay). BSO alone did not induce apoptosis.Conclusion: We conclude that exposure to high oxygen in combination with limited antioxidant protection is responsible for increased cell death via apoptosis in undifferentiated neurons. This finding may contribute to longterm cognitive deficits in preterm infants exposed to high oxygen.

Development of α2u-Globulin Nephropathy and Adrenal Medullary Pheochromocytomas in Male Rats Following Exposure to Stoddard Solvent IIC

Stoddard solvent IIC is widely used as a solvent in paints and varnishes, and for dry cleaning and other grease removal applications. Because concern exists regarding the long-term effects of occupational exposure in industrial settings, the toxicity and carcinogenicity of Stoddard solvent IIC were evaluated in male and female F344/N rats and B6C3F1 mice. Rats and mice were exposed to 0, 138, 275, 550, 1100, or 2200 mg/m3 Stoddard solvent IIC by whole-body inhalation for 3 mo, and to 0, 138 (male rats), 550, 1100, or 2200 (female rats and male and female mice) mg/m3 for 2 yr. The kidney, liver, and adrenal medulla were targets of Stoddard solvent IIC toxicity in rats. After 3 mo of exposure, male rats developed lesions characteristic of α2u-globulin nephropathy. Male and female rats displayed increased liver weights and/or clinical pathology changes suggestive of hepatic injury, although no accompanying histopathologic changes were observed. After 2 yr, increased incidences of adrenal medullary pheochromocytomas provided some evidence of carcinogenicity in male rats. Renal tubule adenomas were slightly increased in male rats after 2 yr, and may have been related to exposure. In mice, there was no chemical-related toxicity after 3 mo, with the exception of increased liver weights in male mice exposed to 2200 mg/m3. After 2 yr, the incidences of hepatocellular adenomas were increased in female mice exposed to 2200 mg/m3; however, these increases were marginal and associated with increases in body weight. There was no evidence of Stoddard solvent IIC carcinogenicity in female rats or male mice. In summary, inhalation exposures of Stoddard solvent IIC resulted in renal toxicity and adrenal medullary pheochromocytomas in male rats. The liver also appeared to be a site of toxicity in male and female rats and mice.

Location of adrenal medullary pheochromocytoma by I-123 metaiodobenzylguanidine SPECT.

This is a retrospective study evaluating the efficacy of SPECT in the location of pheochromocytoma. Thirty patients with a suspected pheochromocytoma underwent I-123 metaiodobenzylguanidine (I-123 MIBG) SPECT 4 and 22 hours after intravenous injection of 370 MBq I-123 MIBG. SPECT was compared with planar scintigraphy, CT scanning, histology, and clinical course. Twenty-two-hour I-123 MIBG SPECT correctly identified 10 patients with adrenal medullary pheochromocytoma and correctly excluded pheochromocytoma in 19 patients. The sensitivity of the 22-hour MIBG SPECT was 1.00 and the specificity was 0.95. The positive predictive value was 0.95 and the negative predictive value was 1.00. In 16 patients, planar scintigraphy was compared with SPECT. SPECT located normal adrenal glands and tumors with greater confidence in three dimensions, but the patients with adrenal medullary pheochromocytoma were all correctly identified by planar scintigraphy. The results of SPECT and CT agreed in 29 of 30 patients (96.7%). I-123 MIBG SPECT for the location of pheochromocytoma has a high sensitivity, specificity, and positive and negative predictive values. I-123 MIBG SPECT or CT scanning alone were equally good for locating adrenal medullary pheochromocytoma but the combination of MIBG SPECT and CT makes it possible to distinguish between functioning and nonfunctioning adenomas. I-123 MIBG SPECT may be used alone or in combination with planar scintigraphy when three-dimensional location of a lesion is wanted.

Staurosporine induces neurite outgrowth in neuronal hybrids (PC12EN) lacking NGF receptors.

A novel neuronal model (PC12EN cells), obtained by somatic hybridization of rat adrenal medullary pheochromocytoma (PC12) and bovine adrenal medullary endothelial (BAME) cells, was developed. PC12EN cells maintained numerous neuronal characteristics: they expressed neuronal glycolipid conjugates, synthesized and secreted catecholamines, and responded to differentiative agents with neurite outgrowth. PC12EN lacked receptors for EGF and both the p75 and trk NGF receptors, while FGF receptor expression was maintained. Staurosporine (5-50 nM), but not other members of the K252a family of protein kinase inhibitors, rapidly induced neurite outgrowth in PC12EN, as also found in the parental PC12 cells, but not in BAME cells. Similarly, both acidic and basic FGF (1-100 ng/ml) were neurotropic in PC12EN. In contrast to the mechanism by which FGF promoted neurite outgrowth in PC12EN, the neurotropic effect of staurosporine did not involve activation of established signalling pathways, such as tyrosine phosphorylation of erk (ras pathway) or SNT (a specific target of neuronal differentiation). In addition, staurosporine induced the tyrosine phosphorylation of the focal adhesion kinase p125FAK. However, since the latter effect was also observed with other protein kinase inhibitors of the K252a family, which induced PC12EN cells flattening but no neurite extension, we propose that FAK tyrosine phosphorylation may be related to ubiquitous changes in cell shape. We anticipate that PC12EN neuronal hybrids will become useful models in neuroscience research for evaluating unique cellular signalling mechanisms of novel neurotropic compounds.

Isolation of peptides arising from the specific posttranslational processing of chromogranin A and chromogranin B from human pheochromocytoma tissue

An extract of human adrenal medullary pheochromocytoma tissue was fractionated by gel permeation chromatography, and peptides of major abundance in the approximate molecular mass range 1000–4000 were purified to apparent homogeneity by reverse phase HPLC. Determination of the primary structures of four such peptides demonstrated that they were fragments of either chromogranin A or chromogranin B. The peptide WSKMDQLAKELTAE represents chromogranin A(324–337), the peptide LGELFNPYYDPLQWKSSHFE represents chromogranin B(498–517), the peptide NLARVPKLDL represents chromogranin B(568–577), and the peptide QYDRVAQLDQLLHY (isolated as the N- terminal pyroglutamyl derivative) represents chromogranin B(580–593). Analysis of the nucleotide sequences of cDNAs complementary to human chromogranin A and B messenger RNAs indicates that each of these peptide sequences is flanked by pairs or groups of basic residues, suggesting that these fragments are the products of specific posttranslational processing. In addition, a peptide identified as chromogranin B(496–517) was isolated from extract. This component represents the product of incomplete proteolytic cleavage at the Lys 494-Arg 495-Lys 496-Arg 497 processing site in chromogranin B. A minor component in the extract was identified as chromogranin B(508–517), but this component probably represents an artifact of the extraction procedure arising from the hydrolysis of the acid labile Asp 507-Pro 508 bond. The study has shown that chromogranin A and B in pheochromocytoma tissue function as the precursors of several small peptides that may have a regulatory role.

Comparative toxicity and carcinogenicity of two chlorinated paraffins in [formula omitted] rats and B6C3F1 mice

The toxicity and carcinogenicity of chlorinated paraffins containing C12 with 60% Cl, and C23 with 43% Cl, were assessed in prechronic and 2-year gavage studies using F344 N rats and B6C3F1 mice of both sexes. Single administrations of chlorinated paraffins were nonlethal in rats and mice, but repeated-dose and 2-year studies demonstrated toxic responses that differed with the paraffins. The C23,Cl43% paraffin produced a granulomatous inflammation in the liver of female rats in 13-week studies, while the C12,Cl60% paraffin caused deaths of rats and mice in 16-day studies and marked liver enlargement in 13-week studies. In 2-year studies, the C23,Cl43% paraffin caused hepatic and lymphatic granulomatous inflammation and hyperplasia in both sexes of rats, and was associated with marginal increases in adrenal medullary pheochromocytomas in female rats and hepatocellular neoplasms in female mice and with clear increases in malignant lymphomas in male mice. The C12,Cl60% paraffin caused marked liver enlargement in rats and increased the severity of nephropathy in male rats and the incidence of nephropathy in female rats. C12,Cl60% also caused hepatocellular neoplasms in both sexes of rats and mice: kidney tubular cell adenomas and adenocarcinomas in male rats, thyroid follicular cell neoplasms in female rats and female mice, and a marginal increase in mononuclear cell leukemia in male rats. Thus, the short-chain, heavily chlorinated paraffin appears to have a greater potential for chronic toxicity and carcinogenicity than the longer-chain, lightly chlorinated paraffin. Both paraffins have been reported to be nonmutagenic in bacteria. (National Toxicology Program (1986) Technical Report, NIH Publications 86-2561 and 86-2564).

Induction of fiber outgrowth in PC12 pheochromocytoma cells by a neuronotrophic factor occurring in human tumors.

Human tumors contain one or several factors which induce fiber outgrowth in a clonal cell line of rat adrenal medullary pheochromocytoma. Its action was tested semiquantitatively in cultured PC12 pheochromocytoma cells using conditioned media from cultures of 45 different brain tumors and homogenates of 18 human tumors of brain and non-brain origin. All tumor homogenates investigated and 71% of the conditioned media were active. The stimulation of fiber growth and the morphological appearance of neurites resembled that seen after treatment with the nerve growth factor (NGF). However, experiments with NGF antiserum and with an NGF non-responsive PC12-mutant indicated that the neuronotrophic factor(s) occurring in human tumors are different from NGF. The active factor is heat labile, unstable at pH's below 4, but stable up to pH9. It retains its activity after dialysis.

SHORT‐TERM REGULATION OF CATECHOLAMINE BIOSYNTHESIS IN A NERVE GROWTH FACTOR RESPONSIVE CLONAL LINE OF RAT PHEOCHROMOCYTOMA CELLS

Abstract— A clonal cell line (designated PC12) has been previously established from a transplantable rat adrenal medullary pheochromocytoma. Tissue cultures of PC12 cells synthesize, store, release and take up catecholamines. PC12 cells also respond to nerve growth factor (NGF) protein by cessation of mitosis and extension of neurites. The present studies concern the comparison of several aspects of catecholamine metabolism in PC12 cultures with that in normal noradrenergic tissues. One question was why the ratio of dopamine to norepinephrine in PC12 cultures (in contrast to that in normal noradrenergic tissue) is considerably more than one. The presence of exogenous reduced ascorbate (a cofactor for dopamine‐β‐monooxygenase) enhanced by 5–10‐fold the rate at which PC12 cultures converted [3H]tyrosine to [3H]norepinephrine. Under such conditions, the rate of synthesis of [3H]do‐pamine was unchanged. It was also found that the ratio of norepinephrine to dopamine increased by 10‐fold when the cells were grown in vivo as tumors. Since tissue culture medium is essentially free of reduced ascorbate, it is likely that the absence of this cofactor is responsible for the low norepinephrine to dopamine ratio in PC12 cultures. Experiments were also carried out on short‐term regulation of catecholamine synthesis in PC12 cultures. These studies revealed the following: (1) The rate of conversion of [3H]tyrosine to [3H]catechols was increased 2–3‐fold (as compared with controls) in the presence of depolarizing levels of K+ (51.5 mM), and by 2‐fold in the presence of 0.5–2 mM‐dibutyryl cyclic adenosine 3′, 5’monophosphoric acid (db‐cAMP). (2) Similar increases occurred in cultures which had been treated with (and had responded to) nerve growth factor. (3) The stimulatory effects of 51.5 mM‐K+ rapidly returned toward control levels when the cultures were returned to control medium and (4) required the presence of Ca2+ in the extracellular medium. (5) Stimulation of catechol synthesis by 51.5 mM‐K+ and db‐cAMP also occurred in the presence of an inhibitor of DOPA decar‐boxylase. Thus, the ultimate effects of these agents were probably at the level of conversion of tyrosine to dopa by tyrosine 3‐monooxygenase. (6) Simultaneous exposure of cultures to 51.5 mM‐K+ and mM‐db‐cAMP gave additive levels of stimulation. Such findings demonstrate that catecholamine synthesis in cultures of PC12 cells undergoes short‐term regulation which is similar to that previously demonstrated in normal monoaminergic tissues. As a homogeneous tissue culture line, the PC12 bears certain advantages for studying the primary mechanisms of such effects.

ADRENAL PHEOCHROMOCYTOMA WITH BOTH BENIGN AND MALIGNANT COMPONENTS

A case of adrenal pheochromocytoma is reported characterized by polygonal basophilic granular cells of benign type, plump eosinophilic granular cells of transitional type, and spindle-shaped cells of malignant type. In the primary tumor the neoplastic cells of each type revealed a distinctive topographical distribution. All gradations between the latter two varieties of cells were found, especially in the vicinity of the transitional cell area. Multiple metastases were present in the liver and lungs, where only anaplastic spindle-shaped cells could be found. Ultrastructurally, the benign cells contained predominantly large, rather light, secretory granules with a coarsely granulated core. In the malignant cells, the number, size, and intensity of granules varied considerably from cell to cell or even within a single cell. In general, the malignant cells had a higher frequency of smaller granules with electron-dense homogeneous cores. Moderate amounts of noradrenalin but not significant dopamine or adrenalin could be demonstrated from a metastatic nodule in liver. We postulate that this adrenal medullary pheochromocytoma was benign originally and underwent malignant transformation shortly before the patient's death. This is based upon the patient's clinical features and the peculiar structures of the primary tumor.