Metabolomics Analyses of 14 Classical Neurotransmitters by GC-TOF with LC-MS Illustrates Secretion of 9 Cell-Cell Signaling Molecules from Sympathoadrenal Chromaffin Cells in the Presence of Lithium.

The classical small molecule neurotransmitters are essential for cell-cell signaling in the nervous system for regulation of behaviors and physiological functions. Metabolomics approaches are ideal for quantitative analyses of neurotransmitter profiles but have not yet been achieved for the repertoire of 14 classical neurotransmitters. Therefore, this study developed targeted metabolomics analyses by full scan gas chromatography/time-of-flight mass spectrometry (GC-TOF) and hydrophilic interaction chromatography-QTRAP mass spectrometry (HILIC-MS/MS) operated in positive ionization mode for identification and quantitation of 14 neurotransmitters consisting of acetylcholine, adenosine, anandamide, aspartate, dopamine, epinephrine, GABA, glutamate, glycine, histamine, melatonin, norepinephrine, serine, and serotonin. GC-TOF represents a new metabolomics method for neurotransmitter analyses. Sensitive measurements of 11 neurotransmitters were achieved by GC-TOF, and three neurotransmitters were analyzed by LC-MS/MS (acetylcholine, anandamide, and melatonin). The limits of detection (LOD) and limits of quantitation (LOQ) were assessed for linearity for GC-TOF and LC-MS/MS protocols. In neurotransmitter-containing dense core secretory vesicles of adrenal medulla, known as chromaffin granules (CG), metabolomics measured the concentrations of 9 neurotransmitters consisting of the catecholamines dopamine, norepinephrine, and epinephrine, combined with glutamate, serotonin, adenosine, aspartate, glycine, and serine. The CG neurotransmitters were constitutively secreted from sympathoadrenal chromaffin cells in culture. Nicotine- and KCl-stimulated release of the catecholamines and adenosine. Lithium, a drug used for the treatment of bipolar disorder, decreased the constitutive secretion of dopamine and norepinephrine and decreased nicotine-stimulated secretion of epinephrine. Lithium had no effect on other secreted neurotransmitters. Overall, the newly developed GC-TOF with LC-MS/MS metabolomics methods for analyses of 14 neurotransmitters will benefit investigations of neurotransmitter regulation in biological systems and in human disease conditions related to drug treatments.

[The immunomorphologic analysis of innervation of chromaffin paraganglian cells of the mammalian arteries and heart].

Innervation of chromaffin cells of paraganglia of the wall of mammalian large arterial vessels and heart (in rat, cat, and human) was studied by neuromorphological and immunohistochemical methods. There is established similarity in structure of specialized, "basket"-type nerve endings of the chromaffin cells (ChC) with pericellular nerve apparatuses of sympathetic and parasympathetic autonomic neurons. It is proposed to use immunohistochemical reaction for synaptophysin as method of selective detection of ChC of paraganglia and adrenal medulla. The conclusion is made that synaptophysin-positive terminals (SPPT) found on bodies of ChC and postganglionic neurons represent efferent, rather than afferent, synapses formed by myelinated axons of preganglionic fibers. It is suggested that ChC of paraganglia alongside with their characteristic endocrine function participate in complex mechanisms of chemoreceptor regulation of tissue homeostasis of mammalian blood vessels and heart.

[A rare cause of cardiogenic shock treated by extracorporeal life support: catecholamine secreting paraganglioma]. / Une cause rare de choc cardiogénique réfractaire nécessitant une assistance circulatoire : le paragangliome sécrétant.

Phaeochromocytoma and paraganglioma are chromaffine tumours secreting catecholamines. They are usually revealed by a paroxystic hypertensive crisis associated with headaches, palpitation and sweats. We reported a case of a young patient presenting a state of life threatening cardiogenic shock as unusual revelation of this tumour, requiring the implementation of an extracorporeal life support until myocardial recovery.

Multiple minute nests of incidentally detected paraganglionic cells associated with urothelial carcinoma of the urinary bladder in a 73-year-old woman.

A 73-year-old woman who had undergone resection of urothelial carcinoma (UC) (G3 > G2) of the ureter was also found to have UC (G3) in the urinary bladder, spread throughout the urinary bladder with multiple foci of carcinoma in situ and small papillary cancers. Total cystectomy was therefore performed. On detailed microscopic examination of the extirpated urinary bladder, multiple minute cell nests, 14 in number and less than 2 mm in diameter each, consisting of cells harboring small nuclei and clear cytoplasm, were incidentally detected within the sub-mucosal layer and the proper muscle layer, mainly in the posterior wall of the urinary bladder. Some cell nests were clearly associated with ganglion cells. The cells in minute nests were positive on Grimelius staining and also strongly positive on staining with antibodies to chromogranin A, neuron-specific enolase (NSE), synaptophysin, and vimentin on immunohistochemical staining. In addition, sustentacular cells in the minute cell nests were positive for S100 protein. Staining with antibodies to pancytokeratin AE1/AE3, glial fibrillary acidic protein, and p53 was negative in the cell nests. Based on these findings, the multiple minute foci were diagnosed as paraganglionic cells (PGCs) incidentally detected in the urinary bladder of an elderly woman, in association with UC. Although PGCs are rarely detected in adult human urinary bladder on routine histopathological examination, the possibility of their existence should be kept in mind by pathologists, especially in coexistence with UC. This is the first case of PGCs associated with UC in the human urinary bladder in the English literature.

Peripheral chemoreceptors: postnatal development and cytochemical findings in Sudden Infant Death Syndrome.

The aim of the present study is to give a review of the postnatal development of peripheral chemoreceptors - carotid body, paraganglia, and pulmonary neuroendocrine cells (PNEC) - with implications in Sudden Infant Death Syndrome (SIDS). In the postnatal period, the hypoxic chemosensitivity of the carotid body gradually develops. Changes include proliferation of type I and II cells, increased numbers of dense core vesicles and K+ channels, and modifications of neurotransmitter/neuromodulator and receptor expression. Chromaffin paraganglia show increased expression of nitric oxide synthase and neuropeptides, and increased innervation. Innervation of PNEC develops fully only in the first postnatal period, after which their density falls. The neuropeptides produced by PNEC also changes, with increased expression of calcitonin gene-related peptide and neuropeptide YY and reduced expression of calcitonin and gastrin-releasing peptide. Most of the findings in the carotid body of SIDS victims, i.e., decrease in type I cells and dense cytoplasmic granules, and increase in progenitor cells, indicates immaturity of the carotid body, which may play a role in SIDS in the form of underlying biologic vulnerability. Aorticopulmonary paraganglia hyperplasia and increase of PNEC are also found in SIDS, and may be epiphenomena of alterations of the respiratory function with a pathogenetical role in SIDS. A comprehensive view of the pathogenesis of SIDS should also arise from the integration of peripheral chemoreceptors findings with neuro- and cardiopathologic ones.

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia.

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9+/-1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables--including heart rate (fH), blood pressure, air-breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases--were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2=46.6+/-3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9+/-2.0 to 18.8+/-6.1 nmol L(-1)) and NA (from 7.7+/-2.2 to 19.7+/-6.3 nmol L(-1)) that was accompanied by significant decreases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air-breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25+/-0.04 to 0.49+/-0.07 mmol kg(-1) h(-1)); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L(-1)), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase f(V) during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water-breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.

Epinephrine and dopamine colocalization with norepinephrine in various peripheral tissues: guanethidine effects.

Chemical sympathectomy with guanethidine (Gnt) selectively destroys the postganglionic noradrenergic neurons, whereas dopaminergic fibers and nonneural catecholamine-secreting cells are spared. As a result, the relative proportions of norepinephrine (NE), epinephrine (E), and dopamine (DA) in tissues can be differentially affected. This study was done to show the possible differences in the relative amount of catecholamines in some organs and tissues that might indicate the nature of the secretory cells from which they originate. The contents of NE, E, and DA were assessed in rats neonatally treated with Gnt. Gnt-treated rats showed significantly lower levels of NE (P < 0.01) in all tissues except the adrenal gland and paraganglia. Epinephrine was present in all tissues with mean levels below 25 ng/g, with the exception of the adrenal gland (700 microg/gland) and paraganglia (100 ng/g). Only the heart showed lower values in Gnt-treated rats. Mean DA levels were also very high in paraganglia (530 ng/g). In the Gnt-treated rats, DA levels fell practically to zero except in the duodenum, mesentery, and adrenal, whereas there were high levels in the paraganglia, which were significantly different from controls. The results suggest that the three catecholamines are contained mainly in noradrenergic sympathetic fibers of muscle, white adipose tissue, heart, liver, pancreas, and spleen. The duodenum and mesentery may have dopaminergic fibers or E- and DA-containing nonneural cells. Hepatic-vagus paraganglia contain all the catecholamines in relatively high amounts in nonneural cells, and Gnt treatment raises DA levels without affecting the other amines.

Neonatal catecholamine content of adrenal and extra-adrenal chromaffin tissue after prenatal exposure to dexamethasone.

We investigated the effects of prenatal exposure to dexamethasone on paraganglia and adrenal catecholamine stores in rabbit neonates. We compared pregnant rabbits injected with 0.01 mg x kg(-1) of dexamethasone (Dex) from day 24 to day 27 of gestation to an untreated group of unmanipulated rabbits. A group injected with 0.9% saline solution was added to evaluate the effect of injection and handling. Catecholamines were assessed by HPLC in offspring paraganglia and adrenal glands on days 0, 1, and 7 after birth. Data were analyzed by a two-factor ANOVA and Bonferroni-Dunn and t tests. Statistical significance was accepted at p < 0.05. Paraganglia catecholamine levels were significantly higher in the Dex animals than in the untreated ones at every maturational stage studied. For saline animals, the levels were lower than in the Dex group and higher than in the untreated one. In adrenal glands, the same pattern was observed for noradrenaline only. These findings suggest that such a treatment has a positive long-term effect on catecholamine levels of both structures with a more marked effect on paraganglia, an extra-adrenal structure exerting a main function during the perinatal period in providing the child with catecholamine stores.

Monolayer co-culture of rat heart cells and bovine adrenal chromaffin paraneurons.

This paper describes a method for the preparation of co-cultures of rat heart cells and bovine adrenal chromaffin paraneurons. The most suitable condition for heart cell isolation was when a combination of trypsin-DNAse I in Locke's solution was used for digestion. The best co-culture conditions were obtained when 10(6) heart cells were plated on 7- to 8-d-old adrenal chromaffin paraneuron cultures containing 0.5 x 10(6) cells per 35-mm diameter culture dishes. Measurements of DNA (heart cells and chromaffin paraneurons), monitoring of beating frequency (heart cells), and catecholamine (chromaffin paraneurons) levels and release indicated that both cell types remain viable and functional for several weeks. Heart cells started their characteristic contractile activity 24 h earlier when plated either on viable or lysed chromaffin paraneurons, an effect apparently due to faster surface adhesion of heart cells. The beating frequency of heart cells increased after treatment of co-cultures with either noradrenaline or nicotine, with the latter agent acting indirectly through the release of chromaffin paraneuron catecholamines. Propranolol produced a dose-related inhibition of the responses to either noradrenaline or nicotine, thus suggesting that the increase in myocyte's beating activity was mediated through beta-receptors. Anti-myosin and anti-dopamine-beta-hydroxylase immunostaining was used for cell type identification and for the demonstration of body-to-body and process-to-process contacts between adrenal chromaffin paraneurons and heart cells. This co-culture system will serve as a starting point of further studies directed to understand a) the influence of a cell type on the development and on the phenotypic characteristics of a second cell type and b) the interaction of cells derived from different organs and species.

Postnatal development of catecholamines and response to hypoxia in adrenals and paraganglia of rabbits.

The postnatal development of catecholamine levels in adrenals and paraganglia in newborn rabbits has been analyzed. At birth, the dominant catecholamine was noradrenaline, contributing 66% of the total catecholamine pool at day 1, 55% of which came from the paraganglia. There was a rapid postnatal increase of adrenaline, which constituted 67% of the total catecholamines at day 6 and 97% in the adult. After hypoxia at day 1, the noradrenaline levels decreased in paraganglia but not in adrenals, while adrenaline levels did not significantly change in either organ.

Light- and electron-microscopic demonstration of enkephalin-like immunoreactivity in paraganglia of the human urinary bladder.

The distribution of enkephalin-like immunoreactivity in paraganglia of the urinary bladder of adult humans was studied by use of immuno-electron microscopy. All paraganglionic cells were positively stained. Enkephalin-like immunoreactivity was located in chromaffin granules. Chromaffin cells in the paraganglia showed only few degenerative features, suggesting undisturbed function of the cells.

The effect of aging on extra-adrenal catecholamine storing cells of the rat.

Cell number and catecholamine histofluorescence were determined in three extra-adrenal chromaffin tissues: the abdominal paraganglia, the carotid body and SIF (small intensely fluorescent)-cells, of male Fischer-344 rats at different ages. Catecholamines were demonstrated using the formaldehyde-induced fluorescence method. The number of paraganglia cells in the retroperitoneal area increased 13.6 fold between 3 and 33 months of age, and the volume of abdominal paraganglia approached 65% of the volume of one adrenal medulla. The emission color of some paraganglia cells shifted from greenish-yellow to yellowish-brown with aging, but differences were not observed in cell number of fluorophore color in the carotid body, a chemoreceptor type of paraganglion. The number of SIF cells (an intraganglionic variant of extra-adrenal chromaffin tissue) in the hypogastric ganglion increased significantly between 3 and 33 months. The pronounced increase of the volume of the paraganglia in aged rats may contribute to elevated concentrations of circulating catecholamines in these animals.

The distribution and endocrine nature of the abdominal paraganglia of adult man.

The paraganglia of adult man were studied using the formaldehyde-induced fluorescence (FIF) method for histochemical characterization of biogenic monoamines. Microspectrofluorimetry was used to record the emission spectra and fluorescence intensities of the paraganglionic cells. The study of samples from six patients showed that well vascularized paraganglia were widely distributed throughout the retroperitoneal spaces. The paraganglia exhibited strong FIF with the spectral characteristics of monamines. Treatment with HC1 caused an increase in the fluorescence intensity of the paraganglia and a simultaneous shift of the emission maximum from 480--495 nm. This change suggests the presence of high concentrations of tryptophyl-containing peptides and is not due to monoamines. The possibility of a dual endocrine function for the paraganglia is discussed.

[Ultrastructural organization of chromaffin paraganglia in the ganglia of the solar plexus]. / Ul'trastrukturnaia organizatsiia khromaffinnykh paragangliev v uzlakh solnechnogo spleteniia

Under study was the ultrastructural organization of chromaffin cells in the nodes of the solar plexus of albino rats. The chromaffin cells form accumulations--paraganglia, bent for capillaries. The chromaffin cells are of small size and have a dense glial capsule. The cytoplasms are rich in mitochondria, Golgi Complexes and vesicles containing catecholamines. The state of cytoplasmic components speaks of asynchronous secretory activity of chromaffin cells. Among catecholamine-containing vesicles three main types can be distinguished, different is size, structure of the content and localization in the perikaryon. Excretion of catecholamines in realized through cytoplasmic processes which, getting free of the glial capsule, come out directly to capillaries. The coordinating influence of the sympathetic nervous system on the endocrinous function of chromaffin paraganglia of the solar plexus is exercised by paraganglionic fibres, whose terminal portions form synaptic contacts on the chromaffin cells.

Monoamine-storing cells in the avian aortic wall.

Using the Falck-Hillarp method, monoamine-storing cells were demonstrated in the avian thoracic aorta just above the openings of the ductus arteriosus. By electron microscopy, numerous dense-cored vesicles, 90-220 nm in diameter, were seen in the cytoplasm. The electron opacity of these dense-cored vesicles was reduced when reserpine (5 mg/kg) was administered. These monoamine-storing cells formed typical synapses with the Schwann cell-enclosed nerve terminals. The monoamine-storing cells first appeared in the aortic wall at 9 days in ovo but it remained obscure whether they originated from the neuroectoderm.

The paraganglia, a persisting endocrine system in man.

The paraganglia have been traced in surgical samples of para-aortic and pelvic retroperitoneal tissues using the formal-dehyde-induced fluorescence method for catecholamines. Clusters of catecholamine-storing cells were found in all (12) patients studied. Microspectrofluorometric recordings showed high amounts of catecholamine in the well-vascularized cell groups. A large proportion of the paraganglia, which are most prominent during the fetal period, also persist during postnatal life.

Effects of guanethidine on paraganglionic cells in the superior cervical ganglion of the rat.

Paraganglionic cells in the rat superior cervical ganglion were investigated by fluorescence and electron microscopy following treatment with guanethidine for 5-30 days. Control animals received saline and guanidine. Fluorescence cytophotometric measurements revealed a general decrease in the catecholamine content of paraganglionic cells in guanethidine-treated animals. However a few cell clusters showed focal increases. Similarly by electron microscopy there was a general decrease of cell clusters showing increases. -- guanethidine -- as well as guanidine--treated animals showed non-specific cytological alterations such as mitochondrial swelling and increase of cytoplasmic glycogen. However no changes of catecholamine contents and of dense core vesicles were noted in control animals. These results confirm the conception that in rat paraganglionic cells the dense core vesicles are the main storage site of catecholamines. The marked difference in the response of some cell culsters to the experimental treatment can be considered as evidence of functional heterogeneity of this cell population in the rat superior cervical ganglion.