Feocromocitoma intratorácico: relato de caso / Intrathoracic pheochromocytoma: a case report

Os autores apresentam um caso de feocromocitoma intratorácico demonstrado e localizado através da cintilografia com MIBG-131, e sua correlaçäo com a radiografia convencional e tomografia computadorizada

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A.

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.

cAMP-dependent transcription of the human CYP21B (P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element.

By utilizing chimeric genes constructed from 5'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.

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.

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.

Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Tetrasialoganglioside GO1b reactive monoclonal antibodies: their characterization and application for quantification of GQ1b in some cell lines of neuronal and adrenal origin(s).

Seven monoclonal antibodies (MAbs) directed to tetrasialoganglioside (GQ1b) were established, purified GQ1b being used for immunization and hybridoma screening. All of the MAbs reacted strongly with GQ1b, although they also reacted with other gangliosides, with different specificities and reactivities. Some MAbs (1H10, 2C7, and 3F4) reacted with GD3, GT1a, GQ1b, and GP1c. MAb 1H4 showed broad specificity. It reacted with GD3, GD1b, GD2, GT1a, GT1b, GO1b, GQ1c, and GP1c. MAbs 7F5, 4E7, and 4F10 recognized GT1a, GQ1b, and GP1c. MAb 4F10 was more specific for GQ1b than the other MAbs. Using MAb 4F10, we determined, by means of an immunoassay, the quantities of endogenous GQ1b in some neuronal and adrenal cell lines, GOTO (human neuroblastoma), Neuro2a (mouse neuroblastoma), and PC12 (rat pheochromocytoma). PC12 and Neuro2a cells contained at least 5.1 X 10(6) and 3.9 X 10(5) molecules/cell of GQ1b, respectively. In contrast, no GQ1b was detected in GOTO cells, which are known for their specific neuritogenic response to this particular ganglioside when exogenously added.

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.

Augmented enkephalin-immunoreactivity in adrenaline-producing phaeochromocytomas.

Immunohistochemical studies for methionine- and leucine-enkephalin were performed on 26 phaeochromocytomas to elucidate the patho-physiological roles of enkephalins. Positive reactions were seen in all phaeochromocytomas with varying intensities. The location of methionine- and leucine-enkephalin agreed fairly well with each other. Stronger immunostaining was obtained in phaeochromocytomas secreting both adrenalin and noradrenaline than in those secreting predominantly noradrenaline. Paroxysmal hypertension was frequently observed in patients with adrenalin-secreting phaeochromocytomas, especially those with marked enkephalin positivity. Urinary excretion of metanephrine was significantly correlated with enkephalin positivity. These findings show that all phaeochromocytomas retain the ability to produce enkephalins of the adreno-medullary or extra-medullary chromaffin tissues from which they derive. Augmented enkephalin-immunoreactivity in adrenalin-producing phaeochromocytomas may be interpreted as reflecting a close association of enkephalins with adrenalin under physiological conditions.

Genetic evidence for alpha 2-adrenergic receptor subtypes in rat brain, heart and adrenal gland.

A complementary DNA (cDNA) clone - cA2-47 - corresponding to a new alpha 2-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the alpha 2-receptor poly(A+) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5' half of the coding region of this cDNA at 37 degrees C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet alpha 2-receptor genomic probe failed to hybridize to any rat brain mRNAs. Under lower stringency conditions, hybridization of the full-length cDNA, cA2-47, to selected rat tissue poly(A+) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroendocrine features of adrenal pheochromocytomas: histological and immunocytochemical evaluation.

Neuroendocrine features of 30 surgically removed adrenal pheochromocytomas were evaluated combining conventional histochemistry and immunocytology. The reactivity for neuron-specific enolase (NSE), S-100 protein, vasoactive intestinal peptide (VIP), calcitonin and ACTH was tested according to the peroxidase anti-peroxidase (PAP) method using polyclonal antibodies. The neuroendocrine marker NSE was found in all cases. S-100 protein was present in satellite cells in 11 (36.6%) tumors. Rare immunoreactive cells for somatostatin were found in 16 (53.3%), for VIP in 8 (26.6%), for calcitonin in 7 (23%), and for ACTH in 3 (10%) cases. Our results proved the histological and functional heterogeneity of pheochromocytomas. The multiple synthetic activity is their inherent feature as in other neuroendocrine tumors.

Further characterization of the human adrenal-derived P-glycoprotein recognized by monoclonal antibody MRK 16 reacting with only human P-glycoprotein.

This paper describes further characterization of the 170-180-kDa glycoprotein (P-glycoprotein) recognized by the monoclonal antibody MRK 16 in the human adrenal. By electron microscopy, P-glycoprotein was observed in the adrenal cell membranes. However, MRK 16-defined P-glycoprotein was not found in cow, pig, horse, monkey or rabbit adrenal, indicating that MRK 16 recognizes the non-homologous part of P-glycoprotein of various species. Eleven out of 16 adrenal tumors including 4 cases of primary aldosteronism and 7 cases of Cushing syndrome were intensely stained with MRK 16, whereas pheochromocytoma, non-functioning adrenocortical adenoma with no associated increase of serum adrenal-derived hormones and myolipoma of the adrenal were not. Finally, P-glycoprotein-MRK 16-protein A-Sepharose complex derived from human adrenal possessed marked ATPase activity. Taken together, these data suggest that P-glycoprotein may play a physiological role in the human adrenal.

Different opinions on classification of DNA histograms produced from paraffin-embedded tissue.

Flow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin-embedded adrenal adenomas or non-neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.

A review of the histology, ultrastructure, immunohistology, and molecular biology of extra-adrenal paragangliomas.

This review summarizes our understanding of extra-adrenal paragangliomas, a subject that has evolved considerably during the past several years. Our object was to review the anatomical, histologic, and biological features of normal and neoplastic glands, with emphasis on immunohistologic studies, and briefly discuss the potential application of nucleic acid hybridization. Since it is difficult to predict clinical outcome for patients with paragangliomas, we have emphasized the differences between benign and malignant paragangliomas, concentrating on recent results obtained using immunohistologic techniques. These studies have emphasized the critical importance of the identification, by immunohistologic means, of two distinct cell populations, chief cells (type I) and sustentacular cells (type II). The relationship between these two cell populations, stable in normal glands and benign tumors, is progressively lost in tumors of increasing degrees of malignancy, sustentacular cells being absent from the most progressively metastasizing paragangliomas.

In vivo MR spectroscopic imaging of the adrenal glands: distinction between adenomas and carcinomas larger than 15 mm based on lipid content.

The usefulness of MR spectroscopic imaging for discriminating between lipid and water was applied to the in vivo differentiation of adrenal adenomas from carcinomas. By using the Dixon sequence in 20 patients, the lipid content of 22 adrenal tumors larger than 15 mm was determined. The mean percentage of lipid in 15 adenomas was 13.4% (standard deviation, 8%), compared with 3.5% lipid (standard deviation, 2%) in seven carcinomas. Only one lesion would have been misclassified on the basis of in vivo measurements of lipid content. After surgery, in vitro MR spectroscopy was used to determine the percentage of lipid in excised samples of nine of the 22 tumors. These in vitro measurements confirmed the in vivo results on lesions larger than 20 mm in diameter. Respiratory artifacts appeared to decrease the accuracy of in vivo measurements in smaller lesions. In vivo MR spectroscopic imaging of adrenal tumors appears to be useful for differentiating between adrenal carcinomas and adenomas.

A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.

Production of GAWK (chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value.

GAWK (chromogranin-B 420-493) is a 74 amino acid peptide recently isolated from human pituitaries. Using two different antibodies (directed against GAWK [1-17] and [20-38] fragments) GAWK-LI was measured in tumors from 194 patients and in the plasma of 434 patients by RIA. The highest tissue concentrations of GAWK-LI were found in pheochromocytoma (GAWK [1-17]-LI, 18,173 +/- 3,915; GAWK [20-38]-LI, 17,852 +/- 2,763 [mean +/- SEM] pmol/g wet wt tissue; n = 9), which were at least ten times higher than any other tumors producing GAWK-LI. High concentrations of GAWK-LI were also found in other types of endocrine tumors including carcinoid, medullary carcinoma of thyroid, pancreatic, and ACTH-producing lung tumors. On the other hand, low concentrations of GAWK-LI were found in nonendocrine tumors. Plasma concentrations of GAWK-LI were found to be elevated in patients with endocrine tumor, but more so in those with pancreatic tumors than with pheochromocytomas. Plasma concentrations returned to normal after successful tumor removal. Chromatographic profiles of GAWK-LI in extracts of pheochromocytomas and normal adrenals showed high molecular weight peaks that were absent in the extracts of other endocrine tumors and normal pancreas, suggesting differential tissue-specific processing. Thus GAWK-LI is produced by a variety of endocrine tumors and may serve as a plasma tumor marker, especially in patients with pancreatic endocrine tumors.

Neurokinin B in a human pheochromocytoma measured with a specific radioimmunoassay.

An N-terminally directed antiserum to neurokinin B was raised in rabbits using an immunogen prepared by coupling the free-SH group of neurokinin B extended from its C-terminus by a cysteine residue (NKB-Cys) to an -NH2 group on human serum albumin using a heterobifunctional cross-linking reagent. In radioimmunoassay with 125I-Bolton-Hunter-labelled NKB-Cys as tracer, the antiserum showed no cross-reactivity with other tachykinins. An extract of a human pheochromocytoma, previously shown to contain peptides derived from preprotachykinin A, contained NKB-LI (13 pmol/g wet weight). The retention time of tumor neurokinin on reversed-phase HPLC was the same as that of synthetic neurokinin B. Peptides with the retention times of substance P, neurokinin A, neurokinin A (3-10)-peptide and neuropeptide K were also identified in the tumor extract. NKB-LI was not detected in extracts of a further nine pheochromocytomas or in five carcinoid tumors that expressed the preprotachykinin A gene.

Peptides related to the N-terminus of pro-opiomelanocortin in the human adrenal medulla.

The immunoreactive (IR) human N-terminal (hNT) of pro-opiomelanocortin (POMC) was measured by specific radioimmunoassay (RIA) and characterized by molecular sieving chromatography, concanavalin A-affinity chromatography and reversed-phase high-performance liquid chromatography (HPLC). The IR hNT levels were 380 +/- 144 ng/g wet wt (mean +/- SD) in the adrenal medulla (N:6), 21.6 +/- 6 ng/g wet wt in the adrenal cortex (N:6), and 45.6% ng/g wet wt in pheochromocytoma tissues (N:3). The IR hNT content of the adrenal medulla was found to be at least twice as high as that of the IR ACTH on a molar basis. Molecular sieving chromatography of IR hNT and IR gamma-3-melanotropin (MSH) showed two major molecular forms (apparent molecular weights of 14 and 12 kilodalton). These major forms were also separable using reversed-phase HPLC. In addition, a part of the IR ACTH material from the adrenal medulla extracts was eluted with an apparent molecular weight of 12 kilodalton. This latter form of IR ACTH was also separated from authentic human ACTH (1-39) by HPLC. Results obtained from concanavalin A-agarose chromatography suggest that one part of the IR gamma-3-MSH material from the adrenal medulla might be non-glycosylated. These results indicate the presence of IR hNT and IR gamma-3-MSH-like material in the human adrenal and also suggest a different processing pathway for POMC from that in the pituitary gland.