Opioid peptides and catecholamines in human pheochromocytoma

Opioid peptides (OP) and catecholamines (CA) were measured in twelve human pheochromocytomas (PHEO). In all tumors the CA concentrations were much higher than those OP (range: 300-85,000 fold higher). Large intertumor variability in the levels of both substances was encountered (mean +/- S.E.M. = CA: 44.9 +/- 7.7 mumoles/g; OP: 38.1 +/- 17.1 nmoles/g; range = CA: 10.1-93.1 mumoles/g; OP: 0.7-181 nmoles/g). Norepinephrine (NE) was the main CA in seven of the 12 tumors. In four of these PHEO, NE accounted for 85% or more of the total CA. These "noradrenergic PHEO" derived from the right adrenals, were of smaller size (36 +/- 15g), had the lowest levels of OP (1.1 +/- 0.3 nmoles/g) and CA (28 +/- 10 mumoles/g), produced moderate to severe sustained hypertension (MBP: 160 +/- 11 mmHg) and the most severe and persistent clinical manifestations. Epinephrine (EPI) was the main CA in five of the 12 tumors. These PHEO had intermediate levels of OP (12 +/- 3 nmoles/g), and four of them were of left adrenal origin. Patients bearing these tumors were generally normotensive (MBP: 103 +/- 4 mmHg) and asymptomatic, with occasional paroxysmal crisis. The highest levels of OP (132 +/- 24 nmoles/g) were found in two tumors of extra-adrenal location and in one of right adrenal origin. The proportion of NE and EPI ranged between 60-80% and 20-40% respectively, of total tumor CA. The two extra-adrenal PHEO were the largest of this series (180 and 245g). These patients had mild hypertension (MBP: 118 +/- 7 mmHg) of sustained or paroxysmal course, and frequent symptomatic episodes. Differences in the synthesis, storage, metabolism and release of CA and OP in PHEO probably account for their variable tumoral content, as well as for the clinical heterogeneity produced by these tumors. It remains to be seen whether OP can contribute to the clinical manifestations of patients with pheochromocytomas.     

Integrative analysis of neuroblastoma and pheochromocytoma genomics data

Background

Pheochromocytoma and neuroblastoma are the most common neural crest-derived tumors in adults and children, respectively. We have performed a large-scale in silico analysis of altogether 1784 neuroblastoma and 531 pheochromocytoma samples to establish similarities and differences using analysis of mRNA and microRNA expression, chromosome aberrations and a novel bioinformatics analysis based on cooperative game theory.

Methods

Datasets obtained from Gene Expression Omnibus and ArrayExpress have been subjected to a complex bioinformatics analysis using GeneSpring, Gene Set Enrichment Analysis, Ingenuity Pathway Analysis and own software.

Results

Comparison of neuroblastoma and pheochromocytoma with other tumors revealed the overexpression of genes involved in development of noradrenergic cells. Among these, the significance of paired-like homeobox 2b in pheochromocytoma has not been reported previously. The analysis of similar expression patterns in neuroblastoma and pheochromocytoma revealed the same anti-apoptotic strategies in these tumors. Cancer regulation by stathmin turned out to be the major difference between pheochromocytoma and neuroblastoma. Underexpression of genes involved in neuronal cell-cell interactions was observed in unfavorable neuroblastoma. By the comparison of hypoxia- and Ras-associated pheochromocytoma, we have found that enhanced insulin like growth factor 1 signaling may be responsible for the activation of Src homology 2 domain containing transforming protein 1, the main co-factor of RET. Hypoxia induced factor 1?? and vascular endothelial growth factor signaling included the most prominent gene expression changes between von Hippel-Lindau- and multiple endocrine neoplasia type 2A-associated pheochromocytoma.

Conclusions

These pathways include previously undescribed pathomechanisms of neuroblastoma and pheochromocytoma and associated gene products may serve as diagnostic markers and therapeutic targets.     

Test with clonidine for diagnosing pheochromocytoma]

Noradrenaline, adrenaline and mean arterial pressure prior to and 3 hours after clonidine administration were evaluated in order to assess a value of single dose of 0.3 mg clonidine in the diagnosis of pheochromocytoma. The study involved 12 patients with pheochromocytoma, 17 patients with arterial hypertension, and 9 patients with borderline hypertension. Seven healthy volunteers served as a control group. It was found that clonidine decreased noradrenaline levels in all healthy subjects and patients with the primary blood hypertension and did not affect noradrenaline levels in patients with pheochromocytoma. Clonidine decreased noradrenaline levels in two patients with normal resting noradrenaline levels. Simultaneously, clonidine decreased noradrenaline levels in two patients with normal resting levels of this catecholamine. The obtained results indicate low specificity of the clonidine test and its value in the diagnosis of pheochromocytoma in patients with normal noradrenaline blood levels.     

Anaphylaxis and plasma catecholamines

The concentrations of plasma catecholamines, cyclic AMP glucose, lactate and glycerol were measured in a high IgE-producing strain of Hooded-Lister rats. Immobilization caused an increase in the levels of plasma catecholamines, especially adrenalin. In rats immunized against egg albumin the rise was higher than in non-sensitized controls, possibly indicating a relationship between sympatho-adrenal activity and serum IgE levels. Anaphylaxis caused by egg albumin induced a rapid and huge increase of plasma catecholamines especially adrenaline. The increase of plasma catecholamines was more rapid and pronounced in awake than in anesthetized rats. The plasma levels of adrenaline following anaphylaxis were larger than those obtained following administration of 5 μg adrenaline i.v. Plasma cyclic AMP, glucose and lactate levels were markedly enhanced already before the injection of antigen. On the other hand, glycerol levels were initially low but increased in parallel to the rise in plasma catecholamines. Administration of dextran to Sprague-Dawley rats induced an anaphylactoid reaction and high plasma levels of adrenaline and noradrenaline. The results indicate a massive sympatho-adrenal activation in anaphylactic and anaphylactoid shock and the use of exogenous adrenaline may cause little additional activation of adrenoceptors.     

Various sympathetic derived human tumors differ in neurofilament expression. Use in diagnosis of neuroblastoma, ganglioneuroblastoma and pheochromocytoma

We have extended our analysis of human tumors using antibodies specific for each of the five types of intermediate filaments to neuroblastoma, ganglioneuroblastoma, pheochromocytoma, ependymoblastoma, and alveolar soft part sarcoma. Tumor cells in the three cases of neuroblastoma, as well as in the single case of alveolar soft part sarcoma, did not react positively with sera directed against any of the five intermediate filament types. We suppose, therefore, that neuroblastoma at least may be derived from a cell type - possibly present in peripheral neurones - which in vivo has very few or no intermediate filaments. In ganglioneuroblastoma and in pheochromocytoma the tumor cells were positive when tested with antibodies directed against neurofilaments and negative with those directed against other intermediate filament types. The ependymoblastoma was positive when tested with antibodies directed against glial fibrillary acidic protein (GFA) and negative when tested with antibodies against other intermediate filament types. Use of antibodies to the different intermediate filament types appears to be a valid way in which to classify tumors, and so far the data presented here and elsewhere support the hypothesis that tumor cells retain the intermediate filament type typical of their cell of origin. Wider use of these sera would seem particularly useful in cases such as neuroblastoma, rhabdomyosarcoma or lymphoma where diagnosis is currently difficult using conventional histological stains.     

Catecholamines in plasma and erythrocytes of rats with transplantable pheochromocytoma

Accumulation of catecholamines in erythrocytes (RBC) was compared to rising plasma levels of catecholamines at weekly intervals following transplantation of pheochromocytoma (line P-259) in the New England Deaconess Hospital rat strain. Additionally changes were investigated during a 12 hour interval after tumor was established in PHEO rats. Starting 2 weeks after tumor implant, the concentrations of norepinephrine (NE) and dopamine (DA) in RBC paralleled and correlated strongly with rising levels of plasma NE and DA which were maximum by 4 weeks. Four to 6 weeks after implant, the RBC to plasma (L/P) concentration ratio of NE was 30% higher in PHEO rats than controls (p less than 0.05) indicating a shift in distribution of NE between the 2 circulating pools. Three measurements, 6 hours apart, showed that mean arterial pressure, plasma and RBC NE and DA concentrations were highest in AM in both PHEO and control groups. Shifts in DA were smaller and did not rise in PM as did NE suggesting DA may reflect tumor secretion and NE, tumor secretion plus sympathetic neuronal activity.     

Usefulness of determining conjugated amine catecholamines in blood platelets for diagnosis of pheochromocytoma]

The studies included 14 patients with pheochromocytoma (mean age 39.5 years), 32 patients with arterial hypertension (mean age 39.5 years), and 9 healthy volunteers (mean age 39.5 years). Free and conjugated noradrenaline and adrenaline in blood platelets have been assayed with RIA technique. It was shown that mean concentrations of conjugated noradrenaline and adrenaline in blood platelets are significantly higher in patients with pheochromocytoma than those in hypertensive patients and healthy individuals. However, such test may be used with limitations as there is high percentage of increased values in patients with the primary arterial hypertension. A decreased noradrenaline inactivation in blood platelets of patients with pheochromocytoma has also been observed. This may exert some effect on the diversified clinical course of this type of arterial hypertension.     

Effect of isradipine on urinary excretion of catecholamines and their metabolites in patients with pheochromocytoma]

An effect of calcium antagonist-isradipine-on catecholamines and their metabolites excretion with the urine in 4 patients with pheochromocytoma has been analysed. It was found that the excretion of OFFnoradrenaline and vanilaminomandelic acid is reduced. It may indicate inhibitory effect of calcium antagonists on catecholamines secretion in patients with pheochromocytoma.     

Alternating lateralization of plasma catecholamines and nasal patency in humans

The nose receives both sympathetic and parasympathetic innervation that is manifested by the alternating dominance of sympathetic activity on one side with concurrent parasympathetic dominance on the other. This ultradian rhythm of autonomic function, known as the nasal cycle, averages 2-3 hours in length. Previous experiments have shown that the nasal cycle is correlated in an inversely coupled fashion to the alternating dominance of activity in the two cerebral hemispheres, suggesting a common mechanism of regulation. Here we show that there is an alternation in catecholamine levels of blood drawn from anticubital veins that may also correlate with the nasal cycle. Radioenzymatic measurement of norepinephrine, epinephrine, and dopamine in blood sampled simultaneously from both arms every 7.5 minutes for periods of 3-6 hours demonstrated alternating high levels of catecholamine in one of the two arms. This alternating lateralization of neurotransmitters was observed in 7 out of 7 experiments using resting human male subjects. The ratio of norepinephrine in the two arms also parallels the pattern of airflow in the nasal cycle. This study suggests that the autonomic nervous system may alternate in activity through paired structures.     

Differential secretion of opioid peptides and catecholamines from cultured cells of a human pheochromocytoma tumor

Dissociated cells from a human pheochromocytoma tumor were maintained in culture, and the secretion of opioid peptides (OP), endogenous catecholamines (CA) and preloaded [³H] norepinephrine from these cells was examined. Nicotine, veratridine, barium or Ionomycin stimulated the secretion of OP, endogenous CA and ³H from the pheochromocytoma cells. In general, the different secretagogues were more potent in releasing OP than endogenous CA; ³H secretion was intermediate. Secretion of OP was more sensitive to stimulation by the calcium ionophore Ionomycin and by veratridine than was CA secretion. Nicotine-evoked OP secretion was more sensitive to extracellular calcium concentration than was secretion of CA or ³H. In contrast, bovine adrenal chromaffin cells show no such differential secretion of OP and CA in response to Ionomycin stimulation or to nicotine stimulation under conditions of varying extracellular calcium concentration. The results show that human pheochromocytomas secrete OP as well as CA and that there may be heterogeneous storage pools of CA and OP in cultured pheochromocytoma cells.     

Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma

The addition of a commercially available sulfatase to the incubation mixture for the single isotope radioenzymatic assay of the catecholamines permits the simultaneous assay of the catecholamine sulfates in plasma. The sulfatase is compatible with the catechol-0-methyl transferase of the radioenzymatic assay and cleaves the sulfate to form the free catechol. With the multiple enzyme action it is possible to assay total (sulfated plus free) catecholamines and DOPA. A second series of assays performed in the absence of the sulfatase provides results for each of the free catecholamines and DOPA. In a study with ten normotensive male subjects supine free NE was 19% (range of 10–24%) of the total, E was 12% (range 6–16%), DA was 1% (range 0.5–2%) and DOPA was 45% (range 14–71%) of the total. Upon standing, free NE increased an average of 71% from 196±53 pg/ml (supine) to 336±69 pg/ml. Free NE (30%) and free E (18%) comprised a larger proportion of the total plasma NE and E in standing subjects than in the plasma of the supine subjects. Changes in the free levels and in the ratio of free to total levels of DA and DOPA were less than those noted for NE and E. This assay methodology may be useful in demonstrating individual variations in the ability to sulfate endogenous catechols.     

Dexamethasone increases both catecholamines and methionine-enkephalin in cultured bovine adrenal chromaffin cells and human extramedullary pheochromocytoma cells

The effect of dexamethasone on dispersed cells in primary monolayer culture from bovine adrenal medulla and human extramedullary pheochromocytoma was examined by estimating the level of catecholamines (CAs) and Methionine-enkephalin (Met-enk) in the medium and cells. In cultured bovine adrenal chromaffin cells, dexamethasone caused significant increase in Met-enk levels 18 hours after administration. There was no release of Met-enk and CAs in the medium 10 min after administration, although nicotine did cause a significant release of Met-enk and CAs. A dose response increase in the level of CAs and Met-enk in bovine adrenal chromaffin cells was obtained with doses varying between 0 and 10(-6)M dexamethasone 18 hours after administration. In cultured human extramedullary pheochromocytoma cells, dexamethasone significantly increased the levels of norepinephrine and Met-enk in a dose dependent manner 24 hours after administration. These results suggest that dexamethasone does not act as a secretagogue but may be related to the synthesis of Met-enk and CAs.     

Effects of exogenous catecholamines on plasma catecholamines and glucose in the sea lamprey,Petromyzon marinus

Summary The effects of exogenous dopamine (DA), norepinephrine (NE) and epinephrine (E) on endogenous catecholamine (CA) titers and glycemia were studied with a highly specific and sensitive radioenzymatic assay (REA) in cardiac-cannulated, prespawning sea lampreys. Neither DA nor NE had a specific effect on the endogenous titers of the other two CAs, or on glycemia. In contrast, E caused a strong increase of both DA and NE at three different doses, one of which must have been in the physiological ranges. This increase may be due to direct stimulation of E on the NE and DA cells. E also caused hyperglycemia 45 min after the injection; however, this effect occurred only with unphysiologically high doses. An estimation of the disappearance rate of exogenous CAs revealed a mammalian-like speed, ranging from 3–5.5 min.Abbreviations CA catecholamines - DA dopamine - E epinephrine - NE norepinephrine - REA radioenzymatic assay     

Plasma catecholamines and plasma corticosterone following restraint stress in juvenile alligators

Ten juvenile alligators, mean body mass 793 g, hatched from artificially incubated eggs and raised under controlled conditions, were held out of water with their jaws held closed for 48 hr. An initial blood sample was taken and further samples collected at 1, 2, 4, 8, 24, and 48 hr. Epinephrine, norepinephrine, and dopamine were measured in plasma aliquots of 1.5 ml using high pressure liquid chromatography with electrochemical detection. Corticosterone was measured by radioimmunoassay. Plasma glucose was measured using the Trinder method and plasma calcium, cholesterol, and triglycerides were measured in an autoanalyzer. Epinephrine was about 4 ng/ml at the initial bleed, but declined steadily to < 0.4 ng/ml by 24 hr. Norepinephrine was also about 4 ng/ml at the initial bleed, but rose to over 8 ng/ml at 1 hr, and then declined to < 0.2 ng/ml at 24 hr. A second, but smaller increase in plasma norepinephrine was seen at 48 hr. Plasma dopamine was low at the initial bleed (< 0.7 ng/ml), rose to over 8 ng/ml at 1 hr, then declined to < 0.2 ng/ml. Plasma corticosterone rose progressively for the first 4 hr, declined at 8 hr and 24 hr, then rose again at 48 hr. Plasma glucose rose significantly by 24 hr and remained elevated for 48 hr. Plasma calcium increased at 1, 2, and 4 hr then returned to levels not significantly different from the initial sample at 24 and 48 hr. The white blood cells showed changes indicating immune system suppression. By the end of the treatment the hetorophil/lymphocyte ratio increased to 4.7. These results suggest that handling alligators, taking multiple blood samples, and keeping them restrained for more than 8 hr is a severe stress to the animals.