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Aim: Catecholamines, which are physiologically important neurotransmitters and hormones, apparently decrease in the brain and plasma as some species age. Because this observation has engendered controversy, we used mice to investigate whether age-related changes occur in adrenal catecholamine levels and in the expression of catecholamine synthetic enzymes., Methods: Adrenal glands were collected from male C57BL/6NCr mice at the ages of 6, 12 and 24 months. Catecholamines, such as dopamine (DA), noradrenaline (NA) and adrenaline (AD) from those glands, were measured by using a highly sensitive liquid chromatographic method with peroxyoxalate chemiluminescence reaction detection. Tyrosine hydroxylase (TH), dopa decarboxylase, dopamine beta hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression levels were measured by quantitative real-time polymerase chain reaction., Results: Although DA levels in the adrenals of 24-month-old mice were higher than in 6- and 12-month-old mice, the AD content decreased with age. In such mice, the ratio of DA to NA at 24 months was lower than at 12 months, and the ratio of NA to AD content at 24 months was significantly lower than at 6 months. The mRNA expression ratios in TH, DBH and PNMT in 24-month-old mice were all lower than in 12-month-old mice., Conclusions: These results strongly suggest that catecholamine synthesis, in general, declines with aging in the adrenal glands of mice and that AD, in particular, undergoes a significant decrease with advancing age., (© 2012 Japan Geriatrics Society.)

Rat brain synaptic vesicles (SVs) isolated by gel filtration on Sephacryl S-500 had little Mg2+(H+)-ATPase activity, though it was identified by Western blots with antibodies against the H+-ATPase A-subunit and other vesicle proteins. In contrast, tyrosine hydroxylase and dopa decarboxylase activities in the SVs were substantial, suggesting that the absence of Mg2+(H+)-ATPase activity was not due to inactivation during isolation but rather to the nature of the SVs. The vesicle component reactive to H+-ATPase antibody was also identified in the synaptosomal cytosol, so the antibody for the A-subunit seemed unnecessary to detect H+-ATPase. The SVs contained beta-amyloid precursor protein of approximately 100 kDa. Based on these observations, SVs without Mg2+(H+)-ATPase seemed to play a role(s) in the delivery of cytoplasmic and plasma membrane proteins to nerve terminals as well as in neurotransmission.

The binding of three tracers for monoaminergic terminals was mapped in the brain of healthy young (N=6) and healthy old rhesus monkeys (N=4), aged monkeys with mild unilateral intracarotid MPTP lesions (N=3), and monkeys of intermediate age with severe systemic MPTP lesions (N=6). The ligand for monoaminergic vesicles (+)-[(11)C]dihydrotetrabenazine (+DTBZ) had a mean binding potential (pB) of 1.4 in striatum of the healthy young monkeys, which was reduced by 20% in putamen of the old monkeys. The catecholamine transporter ligand (+)-[(11)C]methylphenidate (+MP) had a mean pB of 1.3 in striatum of the young monkeys, which was reduced by 40% in caudate and putamen of the old monkeys. The DOPA decarboxylase substrate [(18)F]fluoro-l-DOPA (FDOPA) had a mean decarboxylation coefficient (k(3)(S)) of 0.4 h(-1) in striatum of the young group, and was not significantly reduced in the aged group. Of the three ligands, only +DTBZ pB was significantly reduced in striatum of the small group of animals with mild unilateral lesions. In the group with systemic MPTP lesions, the mean reduction of the binding of the three ligands was 80% in the caudate and putamen. However, the decline in +MP pB in the ventral striatum (-75%) exceeded the declines of +DTBZ pB and FDOPA k(3)(S) in that region (-65%), suggesting that compensatory down-modulation of uptake sites may occur in the striatal regions with the least dopamine depletion. Binding of all three ligands was reduced by 50% in the anterior cingulate cortex and in the thalamus, suggesting toxicity of MPTP for extrastriatal catecholamine innervations. +DTBZ binding in the hypothalamus, presumably mainly in serotonin fibers, was unaffected by systemic MPTP treatment. Of the three tracers, +DTBZ was most sensitive for detecting MPTP-induced dopamine depletion in monkey striatum.

New metabolic diseases are regularly identified by a genetic or biochemical approach. Indeed, the metabolic diseases result from an enzymatic block with accumulation of a metabolite upstream to the block and deficit of a metabolite downstream. The characterization of these abnormal metabolites by MRI spectroscopy permitted to identify the deficient enzyme in two new groups of diseases, creatine deficiencies and polyol anomalies. Creatine deficiency is implicated in unspecific mental retardation. A low peak of creatine at MRI spectroscopy is evocating of creatine deficiency which is treatable by creatine administration. Deficiency of synthesis of polyols, metabolites on the pentose pathway, represent new described metabolic diseases with variable symptoms including a neurological distress, liver disease, splenomegaly, cutis laxa and renal insufficiency. The deficit of ribose-5-phosphate isomerase, one of the enzymes whose diagnosis is evoked in front of the accumulation of ribitol, arabitol and xylitol leads to a leucodystrophy in adults. This new deficit was highlighted by the identification of an abnormal peak in cerebral MRI-spectroscopy corresponding to the abnormal accumulation of polyols in brain. Congenital hyperinsulinism (HI) is characterized by profound hypoglycaemia related to inappropriate insulin secretion. Focal and diffuse forms of hyperinsulinism share a similar clinical presentation but their treatment is dramatically different. Until recently, preoperative differential diagnosis was based on pancreatic venous sampling, an invasive and technically demanding technique. Positron emission tomography (PET) after injection of [18F]Fluoro-L-DOPA has been evaluated for the preoperative differentiation between focal and diffuse HI, by imaging uptake of radiotracer and the conversion of [18F]Fluoro-L-DOPA into dopamine by DOPA decarboxylase. PET with [18F]Fluoro-L-DOPA has been validated as a reliable test to differentiate diffuse and focal HI and is now a major differential diagnosis tool in infantile hyperinsulinemic hypoglycaemia.

We have examined the utility of DDC as a novel marker for the detection of peritoneal micrometastases of gastric cancer. DDC mRNA in the peritoneal wash from 114 gastric cancer patients was quantified for a comparison of carcinoembryonic antigen (CEA) mRNA by means of real-time RT-PCR with a fluorescently labeled probe to predict peritoneal recurrence. The cut-off value was set at the upper limit of the quantitative value for non-cancer patients, and those above this cut-off value constituted the micrometastasis (MM+) group. Thirteen of 15 cases with peritoneal dissemination were MM+DDC (87% sensitivity), and one of 48 t1 cases was MM+ (98% specificity). DDC levels in peritoneal washes from patients with synchronous peritoneal metastases were more than 50 times higher than in those from patients without metastasis (p<0.01). For 15 cases of peritoneal dissemination (seven cases were cytologically positive), DDC was positive in 13 cases (87% sensitivity), but CEA failed to detect micrometastases in four cases (73% sensitivity), indicating that DDC is in some cases superior to CEA for the detection of peritoneal micrometastases of gastric cancer in terms of sensitivity as well as specificity, especially for poorly differentiated adenocarcinomas. Combination of CEA and DDC improved the accuracy of diagnosis up to 93%. These results suggest that DDC is potentially a novel marker for peritoneal dissemination of gastric cancer and that quantitative RT-PCR of DDC is reliable and efficient for the selection of patients for adjuvant intraperitoneal chemotherapy to prevent peritoneal recurrence.

A highly sensitive molecular method was used to evaluate the presence of dopamine decarboxylase (DDC) mRNA in the bone marrow and peripheral blood of patients with neuroblastoma (NB). DDC, like tyrosine hydroxylase (TH), is an enzyme involved in the catecholamine synthesis pathway and has recently been proposed as a specific marker of NB among pediatric malignancies. DDC transcript was detected in five of five NB cell lines, 10 of 10 NB primary tumors, 17 of 18 (94%) bone marrow samples, and 12 of 18 (66%) blood samples drawn at diagnosis in 18 patients affected by disseminated NB. In contrast, no PCR signal was found in 20 bone marrow samples obtained from patients with other malignancies or in eight of nine marrow and blood samples drawn from patients with localized NB (two stage 2 and seven stage 3). In addition, all marrow and blood samples obtained from NB patients at relapse revealed DDC mRNA. Furthermore, the percentage of DDC-positive samples was lower among the samples drawn from these patients during treatment. By comparison with conventional methods for disease evaluation, DDC transcript research can increase the sensitivity of NB cell detection in marrow and blood samples at diagnosis and during the treatment and follow-up of NB patients. These results suggest that finding DDC mRNA in NB patients could be a potential marker for minimal residual disease study.

The effect of ladasten (50 mg/kg) on the activity of tyrosine hydrolase (TH) and DOPA-decarboxylase (DDC) gene expression and on the content of dopamine and L-DOPA in the striatum and hypothalamus in rat brain was studied depending on the duration of drug action. In the initial stage (first hours) of the drug action, the dopaminergic effects are related to an increase in the dopamine release. The observed accumulation of L-DOPA and dopamine is correlated with the transcription activity of genes studied. This leads to a conclusion that the pharmacological activity of ladasten is related to activation of de novo synthesis of TH and DDC. There is a certain difference in the ladasten action upon the TH and DDC gene expression of the key enzymes in hypothalamus and striatum of rat brain.

We previously performed a global analysis of the gene expression of gastric cancer cell lines established from metastases to the peritoneal cavity with the cDNA microarray method, which made it possible to analyse the expression of approximately 21168 genes for the identification of novel markers for the detection of micrometastases in the peritoneal cavity. One of the upregulated genes is dopa decarboxylase (DDC), which is responsible for the synthesis of the key neurotransmitters dopamine and serotonine. We have examined its potential as a novel marker for the detection of peritoneal micrometastases of gastric cancer.DDC mRNA in the peritoneal wash from 112 gastric cancer patients was quantified for comparison of carcinoembryonic antigen (CEA) mRNA by means of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a fluorescently labelled probe to predict peritoneal recurrence. The quantity of DDC and CEA correlated with wall penetration. Real-time RT-PCR could quantitate 10-10(6) DDC-expressing gastric cancer cells per 10(7) mesothelial cells. The cutoff value was set at the upper limit of the quantitative value for noncancer patients, and those above this cutoff value constituted the micrometastasis (MM+) group. Of 15 cases with peritoneal dissemination, 13 were MM+DDC (87% sensitivity), and one of 48 t1 cases was MM+ (98% specificity). DDC levels in peritoneal washes from patients with synchronous peritoneal metastases were more than 50 times higher than in those from patients without metastasis (P<0.01). For 15 cases of peritoneal dissemination (seven cases were cytologically positive), DDC was positive in 13 cases (87% sensitivity), but CEA failed to detect micrometastases in four cases (73% sensitivity), indicating that DDC is in some cases superior to CEA for the detection of peritoneal micrometastases of gastric cancer in terms of sensitivity as well as specificity, especially for poorly differentiated adenocarcinomas. A combination of CEA and DDC improved the accuracy of diagnosis up to 94%. These results suggest that DDC is potentially a novel marker for peritoneal dissemination of gastric cancer and that quantitative RT-PCR of DDC is reliable and efficient for the selection of patients for adjuvant intraperitoneal chemotherapy to prevent peritoneal recurrence.

The histaminergic system and its relationships to the other aminergic transmitter systems in the brain of the zebrafish were studied by using confocal microscopy and immunohistochemistry on brain whole-mounts and sections. All monoaminergic systems displayed extensive, widespread fiber systems that innervated all major brain areas, often in a complementary manner. The ventrocaudal hypothalamus contained all monoamine neurons except noradrenaline cells. Histamine (HA), tyrosine hydroxylase (TH), and serotonin (5-HT) -containing neurons were all found around the posterior recess (PR) of the caudal hypothalamus. TH- and 5-HT-containing neurons were found in the periventricular cell layer of PR, whereas the HA-containing neurons were in the surrounding cell layer as a distinct boundary. Histaminergic neurons, which send widespread ascending and descending fibers, were all confined to the ventrocaudal hypothalamus. Histaminergic neurons were medium in size (approximately 12 microm) with varicose ascending and descending ipsilateral and contralateral fiber projections. Histamine was stored in vesicles in two types of neurons and fibers. A close relationship between HA fibers and serotonergic raphe neurons and noradrenergic locus coeruleus neurons was evident. Putative synaptic contacts were occasionally detected between HA and TH or 5-HT neurons. These results indicate that reciprocal contacts between monoaminergic systems are abundant and complex. The results also provide evidence of homologies to mammalian systems and allow identification of several previously uncharacterized systems in zebrafish mutants., (Copyright 2001 Wiley-Liss, Inc.)

Recent studies suggest that the initial expression of adrenal phenylethanolamine N-methyltransferase (PNMT) and epinephrine (E) are dependent upon stimulation of adrenal glucocorticoid receptors. However, evidence suggests that the expression of heart and brain PNMT is independent of glucocorticoids. We measured PNMT activity and E levels in adrenal, heart and head over the latter half of gestation in rat fetuses treated chronically with glucocorticoids, and in normal controls. Chronic glucocorticoid treatment ending on embryonic day (e)12 did not affect heart, head or trunk PNMT activity or E levels. In contrast, chronic glucocorticoid exposure ending e19 or e20 resulted in marked increases in both PNMT and E in adrenal, heart and head tissues. The elevation of E in all three tissues was unaffected by maternal adrenalectomy, indicating enhanced fetal E synthesis. In the absence of exogenous glucocorticoid treatment heart PNMT activity peaked on e12, prior to the earliest reported appearance of glucocorticoid receptors. We conclude that expression of PNMT in all three tissues is glucocorticoid independent until the latter part of gestation when it is readily enhanced by glucocorticoids.

Objective: Attention deficit hyperactivity disorder (ADHD) is a highly prevalent childhood psychiatric disorder characterized by impaired attention, excessive motor activity, and impulsivity. Despite extensive investigation of the neuropathophysiology of ADHD by a wide array of methodologies, the neurobiochemical substrate of this disorder is still unknown. Converging evidence, however, suggests a primary role of the dopaminergic system., Method: This study examined the integrity of presynaptic dopaminergic function in children with ADHD through use of positron emission tomography and the tracer [18F]fluorodopa ([18F]DOPA). Accumulation of [18F]DOPA in synaptic terminals, a measure of dopa decarboxylase activity, was quantified in regions rich in dopaminergic innervation, including caudate nucleus, putamen, frontal cortex, and midbrain (i.e., substantia nigra and ventral tegmentum)., Results: Accumulation of [18F]DOPA in the right midbrain was higher by 48% in 10 children with ADHD than in 10 normal children. Despite its magnitude, this difference would not have reached statistical significance if corrected by the Bonferroni test for multiple comparisons. However, [18F]DOPA in the right midbrain was correlated with symptom severity. No other dopamine-rich regions significantly differed between groups., Conclusions: These findings are suggestive of dopaminergic dysfunction at the level of the dopaminergic nuclei in children with ADHD. Abnormality in dopa decarboxylase activity may be primary or secondary to deficits in other functional units of the dopamine pathway (e.g., receptor, uptake transporter, vesicular transporter, degradation enzymes). Efforts toward defining the origin of this abnormality should help delineate mechanisms of midbrain control of attention and motor behavior important for the understanding of the causes and treatment of ADHD.

Background: Cardiac ganglia were originally thought to contain only cholinergic neurons relaying parasympathetic information from preganglionic brain stem neurons to the heart. Accumulating evidence, however, suggests that cardiac ganglia contain a heterogeneous population of neurons that synthesize or respond to several different neurotransmitters and neuropeptides. Reports regarding monoamine and histamine synthesis and neurotransmission within cardiac ganglia, however, present conflicting information or are limited in number. Furthermore, very few studies have examined the neurochemistry of adult human cardiac ganglia. The purpose of this study was, therefore, to determine whether monoamine- and histamine-synthesizing enzymes and neurotransmitters exist within neurons of adult human cardiac ganglia., Methods and Results: Human heart tissue containing cardiac ganglia was obtained during autopsies of patients without cardiovascular pathology. Avidin-biotin complex immunohistochemistry was used to demonstrate tyrosine hydroxylase, L-dopa decarboxylase, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, tryptophan hydroxylase, and histidine decarboxylase immunoreactivity within neurons of cardiac ganglia. Dopamine, norepinephrine, serotonin, and histamine immunoreactivity was also found in ganglionic neurons. Omission or preadsorption of primary antibodies from the antisera and subsequent incubation with cardiac ganglia abolished specific staining in all cases examined., Conclusions: Our results suggest that neurons within cardiac ganglia contain enzymes involved in the synthesis of monoamines and histamine and that they contain dopamine, norepinephrine, serotonin, and histamine immunoreactivity. Our findings suggest a putative role for monoamine and histamine neurotransmission within adult human cardiac ganglia. Additional, functional evidence will be necessary to evaluate what the physiological role of monoamines and histamine may be in neural control of the adult human heart.

During development of the Drosophila nerve cord, neuroblast 7-3 gives rise to a pair of mitotic sister serotonin neurons in each hemisegment. Here we show that the zinc finger gene eagle, which is expressed in neuroblast 7-3, is essential for specifying the fate of serotonin neurons. We find that loss-of-function eagle mutations produce an unusual differential phenotype with respect to the sister serotonin cells and that eagle is necessary for the maintenance of engrailed and zfh-2 expression in the serotonin neurons. We present a model that uniquely identifies all progeny neurons in the neuroblast 7-3 lineage based on the expression of specific molecular markers, position within the nerve cord and the effect of eagle loss-of-function mutations. Although serotonin is an important neurotransmitter conserved throughout the animal kingdom, we show that hypomorphic alleles of eagle can produce viable adults that have a dramatic reduction in the number of serotonin-producing neurons.

To extend initial characterizations of their phylogenetic utility, sequences from the nuclear genes for elongation factor-1 alpha (EF-1 alpha) and dopa decarboxylase (DDC) are tested for phylogenetic concordance with each other and with previous morphological evidence within the giant silk moth tribe Attacini (Lepidoptera: Saturniidae). The sampling of DDC is expanded from the 690 basepairs of previous studies to 1051 basepairs in the current study. All nine attacine genera are sampled. EF-1 alpha and DDC agree in the placement of seven of nine genera, with placement of the other two not in strong conflict. Combination of the gene sequences results in a nearly fully resolved tree that is consistent with EF-1 alpha alone and agrees with morphology in five of eight groups. Conflict between molecules and morphology is confined to deeper-level relationships within Attacini, where node support for the molecular hypotheses, but not the morphological hypotheses, is generally very strong. A strong signal is contributed by synonymous substitutions in both genes, and by nonsynonymous change particularly in DDC. The molecular phylogeny supports a revision of attacine biogeography in that neither East Asian nor New World genera form monophyletic groups.

The aim of this work was to investigate the possible presence of DOPA decarboxylase (DDC) in endocrine cells of adult rat pancreas. Islet peptide hormones (insulin, glucagon, and somatostatin), as well as DDC, were detected immunohistochemically using the double-immunofluorescence technique and specific antibodies. DDC-like immunoreactivity was present in cytoplasmic granules within endocrine cells located at islet peripheries in a distribution consistent with islet localisation of A cells. Moreover, these same cells stained positively with glucagon antibody. As DDC is an enzyme specifically involved in catecholamine synthesis, insular cells must possess the capacity to elaborate this class of hormone at least up to the dopamine-decarboxylation step. Thus, after further metabolic processing either in A cells or elsewhere, endogenously-synthesised islet catecholamines may be released and participate in paracrine regulation of insulin secretion.

We measured the levels of dopamine, tyrosine hydroxylase (TH) protein, and dihydroxyphenylalanine (DOPA) decarboxylase (DDC) protein in the striatum of 10 patients with idiopathic Parkinson's disease (PD) and 23 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The levels of dopamine were markedly reduced (2% of control) in the striatum of the patients with PD, whereas striatal dopamine in the patients with OPCA ranged from normal (> 60% of control) to moderately reduced (20-60% of control) to severely depleted (< 20% of control). Both TH and DDC protein levels were significantly lower than those of the controls in the striatum of all of the patients with PD and in the subgroup of patients with OPCA having severely depleted dopamine. In contradistinction, TH but not DDC protein levels were reduced in those patients with OPCA having moderately reduced dopamine levels. This suggests that in the early stage of nigrostriatal dopamine neurone degeneration, DDC levels may be less susceptible to neurodegenerative influences than is TH synthesis or, alternatively, DDC synthesis may be more aggressively upregulated. Unexpectedly, from the blot immunolabeling analysis an additional DDC-immunoreactive band of slightly lower apparent molecular mass was detected in two of the patients with PD and in 12 of the patients with OPCA. This additional DDC band, which was not present in any of the control subjects, may reflect posttranslational modification(s) of DDC related to the neurodegenerative process.

We attempted to prospectively select individualized chemotherapy for 165 non-small cell lung cancer patients based on in vitro analysis of neuroendocrine (NE) markers and drug sensitivity testing (DST) using fresh tumor. The chemotherapy used for small cell lung cancer (SCLC) was selected when NE marker expression determined by L-dopa decarboxylase assay was documented. Selection of chemotherapy for other patients was guided by DST results using a modified dye exclusion assay when available; otherwise etoposide and cisplatin was administered. A total of 112 of 165 (68%) specimens were assayed for L-dopa decarboxylase and 36 patients (22%) had DST. In vitro data directed management for 27 of 96 (28%) patients given chemotherapy: 6 with NE markers were treated with the SCLC regimen; and 21 (58% of those with DST) received their DST-selected chemotherapy regimen. There were no significant differences in response rate among all 3 treatment arms (P = 0.076). However, response to chemotherapy for the patients treated prospectively with a SCLC regimen was 3 of 6 (50%), marginally better than patients given their DST-selected chemotherapy regimen (2 of 21; 9%; P = 0.056) or those treated with etoposide and cisplatin (10 of 69; 14%; P = 0.061). When patients whose NE markers were identified retrospectively are included, 4 of 9 (44%) responded to administered chemotherapy, compared to 7 of 55 (13%) with no NE markers present (P = 0.04). There were no differences in survival among the three treatment groups. Cisplatin and etoposide comprised the most active regimen in vitro for tumors from 16 of 36 (44%) patients, potentially limiting the benefit of DST since this is often the empiric therapy for non-SCLC. Furthermore, the correlation between in vitro and clinical response is nonsignificant for all drugs tested, highlighting the overall relative resistance of non-SCLC tumors to currently available chemotherapy.

IA-1 is a recently isolated novel complementary DNA which encodes a protein of 510 amino acids that contains both a zinc finger DNA-binding domain and a putative prohormone domain. mRNA expression of IA-1 has been found thus far only in tumors of neuroendocrine origin. In this report we describe the expression of IA-1 mRNA in a panel of 64 human lung cancer cell lines. IA-1 mRNA was detected by Northern blot analysis in 97% (30 of 31) of small cell lung cancer cell lines. In contrast, IA-1 mRNA was detected in only 13% (4 of 30) of non-small cell lung cancer cell lines. Nine of the 30 (30%) expressed either chromogranin A mRNA or produced L-dopa decarboxylase. Four of these 9 (44%) had detectable levels of IA-1 mRNA. In most of the lung cancer cell lines examined, IA-1 showed high concordance with the other neuroendocrine markers, L-dopa decarboxylase, and chromogranin A. The one exception was a variant small cell lung cancer cell line which expressed low or nondetectable levels of L-dopa decarboxylase. IA-1 is a candidate marker of neuroendocrine differentiation of human lung tumors.

We describe, for the first time, the development of a technique for a long-term selective culture of endocrine (PE) cells from the lungs of normal animals. Epithelial cells were isolated from 1-day-old hamster lungs through mechanical and enzymatic dissociation with collagenase type II. Cells were then cultured in HITES medium which contained RPMI 1640, hydrocortisone, insulin, transferrin, estradiol, sodium selenite, and supplemented with 5% fetal bovine serum (FBS), or medium which contained HITES medium supplemented with bovine serum albumin, phosphoethanolamine, arginine vasopressin, bombesin, and 2% FBS (9N). HITES medium, originally developed for establishment and long-term culture of human small cell lung cancer (SCLC) cell lines, allowed propagation of normal hamster PE cells up to 12 months as a mixed floating-attached cell culture. No difference was noted in the results using HITES or 9N. By 3 months, 80% of the cultured cells contained characteristic dense-core (endocrine type) granules. The cultured PE cells also expressed creatine kinase brain isoenzyme, and general NE markers including neuron specific enolase, and amine handling enzyme activity within the range of SCLC cell lines. Moreover, cultured PE cells contained and secreted immunoreactive calcitonin (iCT) which had a molecular profile similar to that of intact hamster lung. This long-term culture technique should markedly assist in elucidating the role of PE cells in health and disease.

The establishment and characterization of 11 human lung cancer cell lines are described in this article. Nine of these cell lines were established over a 5-year period, from 1983 to 1988, from patients treated at the Kingston Regional Cancer Centre. These include eight definite or probable small cell lung cancer (SCLC) lines and one adenocarcinoma line. In addition, two other SCLC cell lines were characterized. All of the lines have been in continuous culture for more than 2 years. The clinical histories of the patients from whom the cell lines were derived are outlined here. Several features of the cell lines are presented, including the following: (1) a comparison of the histologic features of the cell lines with the original biopsy specimens; (2) the expression of various markers, including cytokeratin, carcinoembryonic antigen, calcitonin, and neuron-specific enolase; (3) activities of the enzymes l-dopa decarboxylase and the brain isoenzyme of creatine kinase; (4) growth characteristics; (5) cloning efficiency in soft agar; (6) tumorigenicity in nude mice; and (7) cytogenetic studies. These cell lines, obtained directly from patients with a spectrum of drug-sensitive and drug-resistant tumors, will be valuable in vitro models of sensitivity and resistance to chemotherapy in lung cancer.

The establishment and characterisation of 7 small cell lung cancer cell lines is described. Four cell lines were established from biopsies taken from untreated patients and one of these was from primary tumour taken via the fibreoptic bronchoscope. The other three were from biopsies taken from patients who had relapsed after chemotherapy. All grow as non-adherent aggregates of cell and express a range of neuroendocrine and epithelial features characteristics of small cell lung cancer cell lines. No relationship was observed between the characteristics of the cell line in vitro and the treatment status of the patient from whom the biopsy was taken. Furthermore, none of the cell lines expressed p-glycoprotein even though 3 were derived from relapse biopsies where chemotherapy included drugs associated with the multidrug resistance phenotype.

Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.

Three ultrastructural cytochemical methods have been used to classify the innervation of the rat renal cortex. Every axon seen contained chromaffin-reactive, dense core vesicles and stained for tyrosine hydroxylase, indicating that they were all catecholaminergic. About 10% of the axons associated with smooth muscle and juxtaglomerular cells of the arteriolar vessels also contained dopa decarboxylase, but this enzyme was not present in any of the peritubular axons. Our results are compatible with the possibility that, in the rat, the juxtaglomerular blood vessels, but not the renal tubules, are supplied by dopaminergic as well as by noradrenergic nerves.

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.

Guinea-pig aromatic-L-amino acid decarboxylase (DOPA decarboxylase, DDC), but not rat DDC, reacts with the antibody against rat histidine decarboxylase (HDC). For determination of the molecular reaction for this cross-reactivity, a cDNA clone of guinea-pig DDC was isolated. Guinea-pig DDC consists of 480 amino acids and its molecular weight is 54,148. The sequence identify of guinea-pig DDC with rat DDC is 86%. Guinea-pig DDC has a region showing 100% sequence identity with rat HDC, but only 67% sequence identity with rat DDC, suggesting that this region is related with the cross-reactivity of guinea-pig DDC and anti-rat HDC antibody.

L-dopa decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-dopa decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-dopa decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-dopa is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-dopa decarboxylase activity has also been tested.

This paper describes a new, inexpensive and highly sensitive assay for aromatic L-amino acid decarboxylase (AADC) activity, using L-5-hydroxytryptophan (L-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. L-5-HTP was used as substrate and D-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from L-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using L-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.

Considerable advances have been made over the past 5 years of our understanding of the biology of lung tumours in particular SCLC. In this chapter, and in other sections of this symposium various aspects of these properties have been discussed. The greater understanding of the properties of these tumours should provide means whereby increased success can be achieved in clinical remissions and long term survival for the majority of patients with these diseases.

The rat kidney contains and excretes more dopamine than can be attributed solely to its originating from intrarenal noradrenergic nerves and plasma-free dopamine. It has recently been suggested that the source of this excess dopamine may be a population of small cells that are present in the renal medulla, and which by fluorescence and electron microscopy appear to contain high concentrations of a monoamine. We have therefore further investigated these cells. After formaldehyde treatment the fluorescence of the cells was characteristic of indoleamines rather than of catecholamine-containing structures. They did not form a visible precipitate with chromium salts in the classic chromaffin reaction. DOPA decarboxylase-like immunoreactivity was present in the adrenal medulla but not in the intrarenal cells or in mesenteric mast cells. 5-hydroxytryptamine (5-HT)-like immunoreactivity was seen in the intrarenal cells and in mesenteric mast cells, but was not evident in adrenal medullary cells. These results indicate that the intrarenal cells are 5-HT-containing mast cells and do not contribute to renal dopamine production.

Increased specific activity of tyrosine hydroxylase (TH) in the brains of embryonic and 29-day-old chickens results from exposure to reserpine during the early stages of embryogenesis. We have also reported significant increases in the steady-state concentrations of catecholamines (CAs) in the brains of these 29-day-old chickens, suggesting permanent alterations in the control mechanisms during a critical period of development. The specificity of embryonic CA depletion in the above findings was examined usind dl-alpha-methyl-para-tyrosine (AMPT) as a depleting agent. AMPT, injected in varying doses into the yolk sac of fertilized chicken eggs prior to incubation, caused embryonic CA depletion by day 10 of embryogenesis but repletion had occurred by 20 days of embryogenesis. TH activity in whole brain and brain parts was elevated in a dose-related fashion at 29 days postnatally by AMPT. However, no changes in steady-state CA levels in whole brain were observed at this time after AMPT. Differences in the responses to reserpine and AMPT are discussed with respect to their pharmacological actions. The data support the contention that early embryonic CA depletion can result in long-term increases in TH activity postnatally, but steady-state levels of product need not necessarily be altered.

High-performance liquid chromatography with electrochemical detection has developed into a tool with excellent capability to monitor picomole amounts of individual metabolites in both tissue and body-fluid specimens. Significant technical advances in the area of microparticle chemically-bonded stationary phases have led to dramatic improvements in both sensitivity and resolution. Reversed-phase systems can be modified to include charged exchange sites by addition of detergents to the mobile phase. Adjustment of the surface charge in this manner permits capacity factors for ionic sample components to be increased or decreased. This concept is quite compatible with electrochemical detection and has provided the foundation for several clinical assays now in routine use. Examples of additional applications are presented for the determination of catecholamines in tissue, 3-methoxy-4-hydroxyphenylglycol in urine, and dopamine-beta-hydroxylase activity in human serum.

The existence of L-histidine decarboxylase (HDC, EC 4.1.1.22)-like immunoreactive (HDC-I) cells in guinea pig retina was demonstrated using antiserum raised against HDC purified from fetal rat liver. The anti-HDC antiserum partially cross-reacted guinea pig L-DOPA decarboxylase (DDC, EC 4.1.1.28), so the histaminergic neurons were carefully identified. Comparison of HDC-I and DDC-like immunoreactive (DDC-I) cell types in adjacent sections revealed that HDC-I structures were found in some horizontal cells and amacrine cells, and double-staining procedures with anti-HDC antiserum and monoclonal anti-DDC antibody showed that HDC-I horizontal cells had no DDC-I structures, but all the HDC-I amacrine cells had DDC-I structures. From the results, some horizontal cells (with HDC-like immunoreactivities but without DDC-like immunoreactivities) were concluded to be histaminergic.

Using a technique by which binding sites for two antibodies can be visualized in single tissue sections, we have studied the distribution of neurones containing DOPA decarboxylase-like and dopamine beta-hydroxylase-like immunoreactivity in ganglia of dog sympathetic chain. Three types of neurones could be distinguished: those that contained both enzymes, and were presumably noradrenergic; those that contained neither enzyme, and were presumably not catecholaminergic; and a group that contained DOPA decarboxylase but lacked dopamine beta-hydroxylase. The numbers of cells of each type were counted in serially-sectioned ganglia from regions of the sympathetic chain thought to contain dopaminergic neurones (T12-L1 and L7-S2). The percentages of total cell numbers contributed by the DOPA decarboxylase-positive, dopamine beta-hydroxylase-negative cells in these regions were similar to the estimates of dopaminergic neurone numbers that can be made from previously obtained biochemical data. Our results are consistent with the presence of dopaminergic neurons in regions of the paravertebral chain supplying the kidney and the distal hindlimb.

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.

The superior cervical ganglion (SCG) in the neonatal mouse and rat has been employed as a model system to study the regulation of ontogeny of presynaptic cholinergic nerves and postsynaptic adrenergic neurons. During postnatal development presynaptic choline acetyltransferase (ChAc) activity increases 30- to 40-fold, whereas postsynaptic tyrosine hydroxylase (T-OH) activity rises 6- to 8-fold. Transection of the presynaptic cholinergic nerves innervating the SCG prevents the normal development of T-OH activity and the normal accumulation of T-OH enzyme molecules in each postsynaptic neuron. The trans-synaptic regulation of T-OH development is apparently mediated by acetylcholine and postsynaptic depolarization, since pharmacologic ganglionic blockade also prevents normal maturation. Ganglion decentralization also prevents the normal maturation of adrenergic nerve terminals, and the development of end-organ innervation by SCG. Consequently, trans-synaptic factors regulate the ontogeny of adrenergic terminals as well as perikarya. Moreover, normal efferent as well as afferent connections are apparently required for sympathetic development, since removal of salivary glands and orbital contents, target organs of the SCG, in neonates also prevents T-OH development in the ganglia. The postsynaptic neuron contributes to the development of presynaptic cholinergic fibers in SCG. Selective destruction of adrenergic neurons in neonatal mice with either 6-hydroxydopamine or antiserum to nerve growth factor prevents the normal maturation of ChAc activity in presynaptic terminals of SCG. Thus, presynaptic and postsynaptic cells appear to exert reciprocal regulatory influences during ontogeny.

We have characterized 14 human colorectal carcinoma cell lines established from primary and metastatic sites by us during the years 1982 to 1985. Five lines were established in fully defined ACL-4 medium and 9 in serum supplemented R10 medium. However, after establishment, cultures could be grown interchangeably in either medium. The lines grew as floating cell aggregates in ACL-4 medium, while most demonstrated substrate adherence in R10 medium. The lines had relatively long doubling times and low cloning efficiencies. Twelve were tumorigenic in athymic nude mice when injected s.c., and two grew i.p. as well. Based on culture, xenograft, and ultrastructural morphologies, the 14 lines could be subtyped as follows: 4 were well differentiated; 5 were moderately differentiated; 4 were poorly differentiated; and 1 was a mucinous carcinoma. Membrane associated antigens characteristic for gastrointestinal cells (carcinoembryonic antigen, CA 19-9, and TAG-72 antigens) were expressed by 50-71% of the lines. Lines expressing carcinoembryonic antigen and CA 19-9 actively secreted these antigens into the supernatant fluids while TAG-72 antigen was not secreted. Surprisingly, 5 of 7 of the original tumor samples tested and 13 of 14 cultured lines expressed L-dopa decarboxylase activity, which is a characteristic enzyme marker of neuroendocrine cells and tumors. In addition, one poorly differentiated cell line contained dense core granules, characteristic of endocrine secretion. Preliminary cytogenetic analyses indicated that 9 of 11 lines examined contained double minute chromosomes. In addition, 3 of the 9 lines with double minutes also had homogeneously staining regions. These findings indicate a high incidence of amplification of one or more as yet unidentified genes.