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Phaeochromocytomas and paragangliomas are rare neuroendocrine tumours. So far, over 20 causative genes have been identified, of which the most frequent and strongest indicator for malignancies are mutations in succinate dehydrogenase subunit B. No curative therapy is available for patients with metastases resulting in poor prognosis. Therapy development has been hindered by lack of suitable model systems. The succinate dehydrogenase complex is located in the inner membrane of the mitochondria and plays a crucial role in the oxidative phosphorylation chain and the tricarboxylic acid-cycle. Succinate dehydrogenase deficiency results in accumulation of the oncometabolite succinate inducing hypoxia inducible factor stabilization, deoxyribonucleic acid and histone methylation inhibition, and impaired production of adenosine triphosphate. It remains unknown which combination of pathways and/or triggers are decisive for metastases development. In this review, the role of mitochondria in malignant succinate dehydrogenase subunit B-associated phaeochromocytomas and paragangliomas and implications for mitochondria as therapeutic target are discussed., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death. Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O 2 ). Hypoxia, at 24h 0.1% O 2 , induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types. N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression. We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The chemopreventive and antineoplastic activities of caffeic acid derivatives are highly dependent on the chemical structures and cancer cell types. The objective of the present study was to investigate the cytotoxicity of bornyl caffeate and the underlying molecular mechanisms in rat pheochromocytoma PC12 cells. Our initial studies demonstrated that bornyl caffeate exhibited potent cytotoxicity in PC12 cells in a concentration- and time-dependent manner. By examining the cell morphology on a fluorescence microscope and detecting the cell surface phosphoserine with Annexin V-FITC, we proposed that bornyl caffeate could induce apoptosis in PC12 cells. We tested this hypothesis by investigating the effects of bornyl caffeate on several apoptosis-related biomarkers. These experiments showed that bornyl caffeate induced the up-regulation of Bax and down-regulation of Bcl-xl, the disruption of mitochondrial membrane potential, the activation of caspase 3 and the cleavage of PARP. Mechanistic studies further revealed that bornyl caffeate caused the depletion of glutathione (GSH), generation of superoxide ion and progressive activation of p38 mitogen-activate protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a concentration-dependent manner. In particular, GSH depletion appeared to be the most important mechanism underlying the cytotoxicity of bornyl caffeate. The preservation of the intracellular GSH contents with N-acetyl-L-cysteine (NAC), GSH and vitamin C abolished the effect of bornyl caffeate on the activation of p38 MAPK and JNK, preserved the integrity of mitochondrial membrane and ultimately rescued the cells from drug-induced cell death. These results suggest that bornyl caffeate induces apoptosis in PC12 cells via stimulating the depletion of GSH, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial transmembrane potential., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Pheochromocytoma and paraganglioma are rare tumors of adrenals as well as the sympathetic and parasympathetic paraganglia. Clinical presentation of these tumors depends on localization, secretory profile and malignant potential. Four distinct syndromes--PGL1-4--are related to mutations in the succinate dehydrogenase gene--mitochondrial complex involved in electron transfer and Krebs cycle. Here we describe etiology, genetics, as well as clinical aspects of SDH-related tumors. We also describe recent discoveries in HIF-related pathway of tumorigenesis and mutations in new SDH-related genes that have improved our understanding of this disease., (Published by Elsevier Ltd.)

Cyclooxygenase-2 (COX-2) plays crucial roles in the development and invasion of tumors. Celecoxib, a selective COX-2 inhibitor, has been shown to be chemopreventive against cancer. However, to date, the mechanisms of these effects remain unclear. In this study, we investigate the effects of celecoxib on voltage-gated calcium channel (VGCC) currents in undifferentiated pheochromocytoma (PC12) cells using whole-cell patch clamp. Our results showed that celecoxib, instead of rofecoxib or NS-398, another selective COX-2 inhibitor, reversibly inhibited the current density of VGCC in a concentration-dependent manner, but had no apparent effects on the cells treated with nifedipine (1 microM), an L-type calcium channel blocker. Upon pre-incubation of PC12 cells with omega-conotoxia GVIA (1 microM), an N-type calcium channel blocker, omega-agatoxin IVA (1microM), a P/Q-type calcium channel blocker, or SNX-482 (1microM), a R-type calcium channel blocker, celecoxib (1microM) inhibited the currents by 36%, 28%, and 25%, respectively. Celecoxib up-shifted the current-voltage (I-V), and hyperpolarizedly shifted the inactivation curve, but did not markedly affect the activation curve. Intracellular application of H89, a protein kinase A inhibitor, failed to affect the celecoxib's VGCC currents inhibition. Taken together, our present results suggested that celecoxib inhibited L-type calcium channels in PC12 cells via a COX-2 independent pathway, which might be responsible for its clinical effects including anti-tumor.

The neurotransmitter norepinephrine is metabolized by monoamine oxidase into an aldehyde intermediate that is further metabolized to the stable glycol derivative, 3,4-dihydroxyphenylglycol (DHPG). In this study, the possible role of aldose reductase in reducing this aldehyde intermediate in human sympathetic neurons has been examined. DHPG is formed when norepinephrine is incubated with aldose reductase in the presence of monoamine oxidase. DHPG metabolism is inhibited by the monoamine oxidase inhibitor, pargyline which prevents the deamination of norepinephrine, and by the aldose reductase inhibitor AL 1576, which inhibits DHPG formation without affecting the deamination of norepinephrine. Although similar formation of DHPG was observed with human liver aldehyde reductase, the production of DHPG was more effective with aldose reductase than aldehyde reductase. Two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase were observed when sympathetic ganglia were chromatofocused. Molecular modeling studies indicate that the energy-minimized structure of 3,4-dihydroxymandelaldehyde bound to aldose reductase is similar to that of glyceraldehyde where the 2'-hydroxyl group forms hydrogen bonds with Trp111 and NADPH. These results suggest that aldose reductase may be important in metabolizing the potentially toxic aldehyde intermediate formed from norepinephrine in human sympathetic ganglia.

Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidase activities were measured in the supernatant (S2) and pellet (P2) fractions obtained by ultracentrifugation of human adrenal tumor preparations. Negligible enzyme activity was found in cortical tumor whereas highly significant activities were present in the P2 fractions of the two pheochromocytoma specimens. The hydrolysis rates, expressed in terms of the percent of added substrate were 58-66%/60 min for Hip-Phe-Arg, 55-58%/60 min for Hip-Arg-Phe and 19-30%/60 min for Hip-His-Leu. The angiotensin-converting enzyme inhibitor, captopril, differentially inhibited the enzyme splitting Hip-His-Leu versus the one cleaving Hip-Arg-Phe; Hip-Phe-Arg is probably the substrate of both. It is concluded that the Hip-Arg-Phe-cleaving enzyme in adrenomedullary tumor is probably identical to the purportedly novel dipeptidyl carboxypeptidase that we detected earlier in rabbit ear artery wall, which converts (Met5)-enkephalin-Arg6,Phe7 to (Met5)-enkephalin.

We performed a comparative study of catecholamine content, tyrosine hydroxylase (TH) activity, and TH mRNA levels in normal human adrenals and various clinical forms of human pheochromocytoma. We studied sporadic, benign intra-adrenal chromaffin tumors and other non-malignant intra-adrenal tumors associated with multiple endocrine neoplasia type 2B (MEN 2B) and von Hippel-Lindau disease along with one extra-adrenal malignant pheochromocytoma. Our findings suggest substantial differences in TH transcriptional rates or the stability of TH mRNA or both may contribute to altered TH expression in human chromaffin cells associated with "normal" adrenal tissues and various forms of pheochromocytoma and distinctive patterns of expression in the different settings in which these tumors arise.

Exposure of PC12-VG cells to an extremely low frequency magnetic field (ELFMF) enhanced the beta-galactosidase gene expression stimulated by treatment of the cells with forskolin. The enhancing effect of the ELFMF was inhibited by treatment of the cells with a specific inhibitor of PKC, calphostin C, as well as with the Ca2+ entry blockers nifedipin and dantrolen. Enhancement appeared within the first hour of a 4h forskolin treatment when the ELFMF was given at different times during culture. We speculate that exposure of PC12-VG cells to an ELFMF during the early response to forskolin treatment affects cell signal transduction, resulting in enhanced gene expression.

Based on the cDNA sequence of the human and bovine endothelin converting enzyme (ECE-1) we have developed a novel reverse transcription polymerase chain reaction to investigate the tissue expression of this gene. We were able to specifically detect the gene mRNA starting from very limited amount of tissue in all human as well as bovine tissues examined. Thus, our results confirm a widespread expression of the ECE-1 gene in human tissues, in keeping with the findings in other species, and suggest a major biological role of ECE-1.

To elucidate the mechanisms of abnormal steroid production in hyperfunctioning and non-hyperfunctioning adrenal tumors, we examined both the activities and amounts of steroidogenic cytochromes P450 in the tumor and non-tumor portions of these adrenals at the posttranslational (protein) level. Adrenals from 5 patients with primary aldosteronism, 5 with Cushing's syndrome, 1 with deoxycorticosterone (DOC)-producing adenoma, 10 with non-hyperfunctioning adrenal adenoma, and 5 subjects with normal control adrenals (obtained from patients with renal cell carcinoma) were used in our studies. Activities of P450scc, P45011 beta and P450aldo, and P450C21 and P-45017 alpha were assayed in a reconstituted enzyme system using 20 alpha-hydroxycholesterol, DOC, and progesterone, respectively, and the substrate and the extracted products were analyzed by HPLC. Enzyme amounts were determined by immunoblot analysis with anti-bovine P450scc, P45011 beta, and P450C21 IgG, and anti-porcine P45017 alpha IgG. Human P450aldo was only detected in the tumor portion of primary aldosteronism adrenals, with both activities and amounts of other P-450s similar to those in the non-tumor portion of primary aldosteronism and normal controls. In Cushing's syndrome, both activities and amounts of P45017 alpha and P450C21 were significantly increased in the tumor compared with those in the non-tumor portion of Cushing's syndrome and normal controls. In DOC-producing adenoma, both activities and amounts of P45017 alpha and P45011 beta in the tumor portion of the adenoma decreased compared with normal control, while those of other P450s were similar to normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of alpha-galactosyltransferase in cultured rat pheochromocytoma subcloned (PC12h) cells was examined using Gb3 as the acceptor for the galactose from UDP-galactose. The major reaction product was identified as gal alpha 1-3Gb3 based on its mobility on thin-layer chromatographic (TLC) plates and susceptibility to specific galactosidases. The enzyme activity in PC12h cells was the highest at pH 7.0 while the presence of Triton CF-54 (0.1%) and Mn2+ (5 mM) was required for its full activity. The apparent Km values for Gb3 and UDP-galactose were 57 and 17 microM, respectively. The enzyme activity in PC12h cells was compared with that in parent PC12 cells, in which gal alpha 1-3Gb3 is not expressed in an appreciable amount. In the enzyme reaction with exogenous Gb3, the enzyme activity in PC12h cells was about 1.5-fold higher than that in PC12 cells. In the absence of exogenous Gb3, this difference became even more pronounced; gal alpha 1-3Gb3 was generated from endogenous Gb3 at a much higher rate in PC12h cells than in PC12 cells. These findings suggest that the higher level of the alpha-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having gal alpha 1-3 terminal residues in the cells.

Rat pheochromocytoma PC 12 cells are susceptible to the oxidative toxicity caused by H2O2, nitrofurantoin, dopamine, and xanthine/xanthine oxidase reaction. The cytotoxicities of these agents are greatly reduced by the simultaneous presence of 0.1 mM tetrahydrobiopterin (BH4), 3 units/ml horseradish peroxidase, 0.2 mM NADH, and 0.1 units/ml sheep liver dihydropteridine reductase (DHPR). Individually, BH4, NADH and DHPR have no protection against H2O2 toxicity in PC 12 cells. Peroxidase alone offers 58% of protection if cells are incubated in the medium but only 3% in Dulbecco's phosphate buffered saline. The efficiency of the BH4-mediated antioxidation system in PC 12 cells is equal to or better than ascorbic acid and catalase, depending on the source of the reactive O2 species (ROS). The reactions responsible for the BH4-antioxidation system may consist of the non-enzymatic and the peroxidase-catalyzed reduction of H2O2 to H2O by BH4 and the regeneration of BH4 by DHPR using NADH as the cofactor. The components of this defence mechanism against ROS are all normal cellular constituents and are ubiquitous in nature. This DHPR-catalyzed redox cycling of BH4 may constitute an as yet little-known antioxidation system in mammalian cells.

The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, showed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.

Twelve hybridomas, which secrete the monoclonal antibodies to rat pheochromocytoma tyrosine hydroxylase (TH), were obtained. All of these antibodies immunoprecipitated the TH molecules from rat pheochromocytoma PC12h cells, adrenal medulla and brain. Two antibodies, namely PCTH-3 and -7 (both IgG1), directly inhibited the catalytic activity of TH. Another antibody, PCTH-4 (IgG1), bound to the enzyme without inhibition of the catalytic activity. The antibodies PCTH-3 and -4 immunostained the 60 K bands on a nitrocellulose sheet, which were electrotransfered from the gel after the SDS-polyacrylamide gel electrophoresis of PC12h cell and rat adrenal medulla homogenates. The antibody PCTH-4 immunocytochemically labeled specific neurons in the locus ceruleus, hypothalamus and substantia nigra of rat brain. Immunotitration using PCTH-4 antibody revealed that the nerve growth factor-mediated increase of TH activity in PC12h cells is due to the increase of the enzyme molecules.

Treatment with neuronal growth factor (NGF) results in the growth of neuronal processes by PC12 cells and a concomitant 70% increase in the area of the Golgi apparatus. To define the observed morphologic changes in biochemical terms, we investigated the effect of NGF treatment on some Golgi and lysosomal enzyme activities of PC12 cells. Enzyme activities characteristic of the Golgi apparatus, lysosomes, plasma membranes, mitochondria, and endoplasmic reticulum were measured in cell homogenates, in post-mitochrondrial supernatants, and in Golgi-enriched fractions from control and from NGF-stimulated PC12 cells. Treatment of PC12 cells with NGF did not change the level of the Golgi activity of UDPGal:GlcNAc galactosyltransferase while that of CMP-sialic acid:lactosylceramide sialyltransferase was increased three- to fivefold in all fractions studied. For lysosomal enzymes, NGF treatment resulted in a two- to threefold higher level of arylsulfatase activity compared to either acid phosphatase or acid alpha-mannosidase activities. These results indicate that there is a selective increase of at least one Golgi and one lysosomal activity as a result of NGF stimulation of PC12 cells. Both of these enzymes are involved in glycolipid metabolism. It is possible that the dramatic morphologic changes observed during NGF-induced differentiation of PC12 cells are associated not only with increased synthesis in the Golgi apparatus of plasma membrane components such as gangliosides, but also with increased degradation in lysosomes of other plasma membrane components such as sulfatide.

Human tyrosine hydroxylase (TH) cDNA was isolated by molecular cloning. Lambda gt 11 cDNA library constructed from human pheochromocytoma was screened with a synthetic 23-mer oligonucleotide complementary to rat TH mRNA. We found a novel type of cDNA clone whose N-terminal sequence is similar to but clearly distinct from each of the three types (type 1, 2 and 3) of TH cDNA reported by Grima et al. [Nature (1987) 326, 707-711]. It contains both the 12-bp insert characteristic of type 2 cDNA and the 81-bp sequence of type 3. This novel cDNA clone was designated as type 4. Southern blot analysis of human genomic DNA indicated that TH is encoded by a single gene. This suggests that the four different forms of TH mRNA are produced by alternative RNA splicing from a single primary transcript.

Tyrosine hydroxylase [E.C. 1.14.16.2] is inactivated by incubation with its reduced pterin cofactors L-erythro-tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deazatetrahydropterin, which has no cofactor activity, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase.

Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7-dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3- to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists.

We have studied the changes in the levels of the enzyme molecule and mRNA for poly(ADP-ribose) synthetase during nerve growth factor-promoted neurite outgrowth in rat pheochromocytoma PC12 cells. When the PC12 cells were cultured in the presence of nerve growth factor, the content of enzyme molecules decreased along with neurite outgrowth to 50% of the original amounts in 2 days and the content of mRNA for the enzyme also decreased to approximately 50% in 2 days. These results suggest that the decrease of the enzyme molecule may be due to depression of expression of the gene for synthetase during the process. Taken together with previous observations, the decrease of the synthetase seems to be required for some cellular differentiation.

Release of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and 125I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background 125I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

Six amine, amino acid and peptide derivatives derived from 1,3-dipropyl-8-(p-carboxymethylphenyl)xanthine, a functionalized congener of 1,3-dipropyl-8-phenylxanthine, have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC 12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. The functionalized congeners and conjugates have affinity constants ranging from 80 to 310 nM at A2 receptors of PC 12 cells and from 25 to 135 nM at those of platelets. The affinity of the xanthine derivatives at A1 receptors of fat cells are in the 15 to 30 nM range. Thus, the amino acid and peptide conjugates have high potencies at both receptor subclasses and show some selectivity toward A1 adenosine receptors. Derivatives of the congeners should be useful as receptor probes and as radioiodinated ligands.

To determine whether pheochromocytoma phenol sulfotransferase (PST) activities were similar to blood platelet PST activities, we established assay conditions and biochemical properties for the human pheochromocytoma enzymes. At least two forms of PST were present in high speed supernatant (HSS) preparations of the tumors. A thermolabile form (TL) and a thermostable form (TS) were similar to those of human platelet PST with regard to pH optima, apparent Km values, responses to 2,6-dichloro-4-nitrophenol and thermal stability. PST activities were measured in 74 tumors of neuroectodermal origin that had been stored at -80 degrees C for a mean of 37.9 months. Levels of TL and TS PST activities decreased in a nonlinear fashion with time of sample storage. TL and TS PST activities of 4 samples assayed after 1.08 +/- 1.95 (mean +/- SD) month of storage were 167 +/- 73 and 3,110 +/- 1,817 U/mg protein, respectively (mean +/- SEM). Our results indicated that the TL and TS forms of PST in pheochromocytoma HSS preparations were biochemically similar to platelet PST activities.

A simple assay procedure for tyrosine hydroxylase activity in crude tissue samples was devised that requires minimal sample preparation and use of high-performance liquid chromatography with coulometric electrochemical detection. After incubation of enzyme samples, such as human brain homogenates or rat pheochromocytoma PC12h cells, with L-tyrosine and a tetrahydropterin cofactor, in the presence or absence of p-bromobenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, the reaction was terminated by addition of an equal volume of 0.1 M perchloric acid. For quantitation of L-DOPA produced, the sample was centrifuged, filtered and directly applied to the chromatographic apparatus connected to a coulometric electrochemical detector. This method makes redundant a time-consuming step in the previous methods, purification and concentration of L-DOPA or dopamine using alumina. The reaction conditions for the assay of tyrosine hydroxylase activity in brain homogenates and PC12h cells were re-examined by this method. Both tyrosine hydroxylase samples required a naturally occurring cofactor, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4], catalase and NSD-1055 for the full activity, and tyrosine hydroxylase in human brain homogenates required Fe2+ ions for its full activity. (6R)BH4 proved to be a more effective cofactor than a synthetic cofactor, (6RS)-methyl-5,6,7,8-tetrahydropterin, which is commonly used for this assay.

In 3 patients with proved pheochromocytoma, circulating catecholamine concentrations, vanillylmandelic acid and total free catecholamine urinary excretion, dopamine-beta-hydroxylase activities in plasma were measured the day before surgery, during the removal of the tumour and postoperatively. Following the removal of the pheochromocytoma, all these parameters declined with a different rate, possibly related to the half-life proper to each molecule. The results showing a decrease in plasma D beta H activities after the removal of the tumors are in good accordance with most authors. The mechanism and the meaning of catecholamines release from non-innervated tumor-cells remain to be elucidated. By considering these three cases, it does not seem that the evolution of D beta H activity after removal of the tumor is dependent upon the fact that the pheochromocytoma releases preferentially either epinephrine or nephinephrine.

Glycogen phosphorylase in PC12 cells exists in two forms analogous to those found in brain and muscle. The active phosphorylated form of the enzyme, phosphorylase-a, represents about 20-30% of total glycogen phosphorylase in these cells. Incubation of PC12 cells with 100 ng 7S nerve growth factor/ml increased phosphorylase-a within minutes. In contrast to nerve growth factor, insulin (6 ng/ml) and epidermal growth factor (6 ng/ml) decreased phosphorylase-a. Activation of phosphorylase-a by nerve growth factor was not accompanied by increases in cyclic AMP; however, removal of extracellular Ca2+ or incubation of cells with calcium channel blockers inhibited activation of glycogen phosphorylase by nerve growth factor.

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.

Aromatic-L-aminoacid (dopa) decarboxylase (ALAAD) was determined in human plasma by its ability to form dopamine from the substrate 3,4-dihydroxyphenylalanine in the presence of pyridoxal-5-phosphate as cofactor. Dopamine formed was quantitated by high performance liquid chromatography with electrochemical detection. A preincubation step of plasma with the cofactor and dithioerythritol was necessary to obtain optimal reaction conditions. The assay method showed good linearity and reproducibility. The inhibition pattern of the therapeutically used peripheral dopa decarboxylase inhibitors, carbidopa and benserazide, was studied and appeared to be dependent on whether the inhibitor was added before or after the preincubation step. Mean levels in 40 control subjects, in 40 patients with essential hypertension and in 15 patients with phaeochromocytoma, were 34.6 (SD 12.1), 28.5 (SD 10.9) and 34.7 (SD 18.4) mU/l respectively. In the patients with essential hypertension the enzyme level decreased with age (p less than 0.05). Very high levels were found in plasma of two patients with metastatic phaeochromocytoma and in two patients with untreated neuroblastoma, but not in two patients with neuroblastoma after chemotherapy. The method described can be used for measuring uninhibited ALAAD activity in patients treated with benserazide, as well as for measuring total, i.e. the sum of inhibited and uninhibited, ALAAD activity in patients treated with carbidopa.

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.

We purified aromatic L-amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an Mr of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated L-3,4-dihydroxyphenylalanine, L-5-hydroxytryptophan, and L-threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.