RESUMEN
In chromaffin cells, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine ß-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) are mainly involved in catecholamine synthesis. In this study, we evaluated the association between the status of catecholamine-synthesizing enzymes and histopathological features of pheochromocytoma and extraadrenal paraganglioma with special emphasis upon their postoperative clinical behavior. Immunohistochemical evaluation of TH, DBH, AADC, PNMT, Ki 67, and S-100 was performed in 29 pheochromocytoma and 10 extraadrenal paraganglioma and one lymph node harboring metastatic pheochromocytoma. Among these cases, metastasis was subsequently developed in three cases. Urinary normetanephrine (U-NM) levels were significantly higher in clinical metastatic cases than non-metastatic ones. Ki 67 labeling index was significantly higher in both clinical metastatic cases and the Adrenal Gland Scaled Score (PASS) score of ⧠4 cases than PASS < 4 cases, although this score was originally used in pheochromocytoma. H-score of AADC and DBH were significantly lower in PASS ⧠4 cases than those with < 4 cases, and in the cases associated with intratumoral necrosis (n = 4), the presence of spindle shaped tumor cells (n = 4), and large nests of cells or diffuse growth (n = 5). Lower status of intratumoral AADC could be related to poor differentiation of tumor cells in both catecholamine production and morphology and could be related to aggressive biological behavior of both pheochromocytoma and extraadrenal paraganglioma.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/enzimología , Catecolaminas/biosíntesis , Paraganglioma Extraadrenal/enzimología , Feocromocitoma/enzimología , Neoplasias de las Glándulas Suprarrenales/patología , Adulto , Descarboxilasas de Aminoácido-L-Aromático/análisis , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Enfermedades del Sistema Nervioso Autónomo/metabolismo , Dopamina beta-Hidroxilasa/análisis , Dopamina beta-Hidroxilasa/deficiencia , Dopamina beta-Hidroxilasa/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Norepinefrina/análisis , Norepinefrina/deficiencia , Norepinefrina/metabolismo , Paraganglioma Extraadrenal/patología , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/metabolismo , Feocromocitoma/patología , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
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.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Envejecimiento/metabolismo , Dopamina/análisis , Epinefrina/análisis , Neurotransmisores/análisis , Norepinefrina/análisis , Glándulas Suprarrenales/enzimología , Envejecimiento/patología , Animales , Peso Corporal , Cromatografía Liquida , Dopa-Decarboxilasa/análisis , Dopamina beta-Hidroxilasa/análisis , Luminiscencia , Sustancias Luminiscentes , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Tamaño de los Órganos , Oxalatos , Feniletanolamina N-Metiltransferasa/análisis , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Tirosina 3-Monooxigenasa/análisisRESUMEN
Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-L-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.
Asunto(s)
Epinefrina/metabolismo , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/metabolismo , Espectrometría de Masas en Tándem/métodos , Neoplasias de las Glándulas Suprarrenales/enzimología , Animales , Antineoplásicos Hormonales/farmacología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Deuterio/análisis , Deuterio/metabolismo , Dexametasona/farmacología , Pruebas de Enzimas/métodos , Epinefrina/análisis , Humanos , Ratones , Norepinefrina/metabolismo , Feocromocitoma/enzimología , S-Adenosilmetionina/metabolismo , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: Social isolation is regarded as one of the most relevant causes of diseases in mammalian species. The activation of the sympathoneural system represents one of the key components of the stress response. The sympathetic nervous system is one of the major pathways involved in immune-neuroendocrine interactions. The aim of the present study was to determine plasma epinephrine and norepinephrine in individually housed rats, as well as to find out whether splenic gene expression of catecholamine synthesizing enzymes and their protein levels are affected by chronic psychosocial stress. METHODS: Tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels were quantified by quantitative real-time RT-PCR. The TH, DBH and PNMT immunoproteins were assayed by Western blot. RESULTS: Chronic social isolation of adult male rats produced a significant increase in plasma catecholamine levels and a decrease in splenic TH mRNA, DBH mRNA and PNMT mRNA. Protein levels of TH, DBH and PNMT were also reduced. CONCLUSION: These results suggest that increased plasma catecholamines and decreased gene expression and protein levels of catecholamine biosynthetic enzymes in the spleen of chronically individually housed animals might reduce catecholamine synthesis, thus leaving the immunocompetent tissues depleted of catecholamines and consequently leading to an impairment of immune response.
Asunto(s)
Catecolaminas/biosíntesis , Neuroinmunomodulación/fisiología , Estrés Psicológico/enzimología , Estrés Psicológico/inmunología , Sistema Nervioso Simpático/enzimología , Sistema Nervioso Simpático/inmunología , Animales , Catecolaminas/sangre , Enfermedad Crónica , Modelos Animales de Enfermedad , Dopamina beta-Hidroxilasa/análisis , Dopamina beta-Hidroxilasa/genética , Dopamina beta-Hidroxilasa/metabolismo , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Regulación de la Expresión Génica/inmunología , Sistema Inmunológico/fisiología , Masculino , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/genética , Feniletanolamina N-Metiltransferasa/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Aislamiento Social/psicología , Bazo/enzimología , Bazo/metabolismo , Estrés Psicológico/fisiopatología , Sistema Nervioso Simpático/metabolismo , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.
Asunto(s)
Fundus Gástrico/inervación , Norepinefrina/metabolismo , Rombencéfalo/ultraestructura , Nervio Vago/ultraestructura , Aferentes Viscerales/ultraestructura , Animales , Área Postrema/fisiología , Área Postrema/ultraestructura , Vías Autónomas/fisiología , Vías Autónomas/ultraestructura , Comunicación Celular/fisiología , Toxina del Cólera , Dendritas/fisiología , Dendritas/ultraestructura , Dopamina beta-Hidroxilasa/análisis , Dopamina beta-Hidroxilasa/metabolismo , Fundus Gástrico/fisiología , Peroxidasa de Rábano Silvestre , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/metabolismo , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Rombencéfalo/fisiología , Sistema Nervioso Simpático/fisiología , Sistema Nervioso Simpático/ultraestructura , Transmisión Sináptica/fisiología , Nervio Vago/fisiología , Aferentes Viscerales/fisiologíaRESUMEN
Phaeochromocytomas are rare neuroendocrine tumours that produce catecholamines and numerous secretory proteins and peptides, including neuropeptide Y (NPY), a vasoactive peptide with influences on blood pressure. The production of catecholamines and NPY by phaeochromocytomas is highly variable. This study examined influences of hereditary factors and differences in catecholamine production on tumour expression of NPY, as assessed by quantitative PCR, enzyme immunoassay and immunohistochemistry. Phaeochromocytomas included hereditary adrenaline-producing tumours (adrenergic phenotype) in multiple endocrine neoplasia type 2 (MEN 2), predominantly noradrenaline-producing tumours (noradrenergic phenotype) in von Hippel-Lindau (VHL) syndrome, and other adrenergic and noradrenergic tumours where there was no clear hereditary syndrome. NPY levels in phaeochromocytomas from VHL patients were lower (P<0.0001) than in those from MEN 2 patients for both mRNA (84-fold difference) and the peptide (99-fold difference). These findings were supported by immunohistochemistry. NPY levels were also lower in VHL tumours than in those where there was no hereditary syndrome. Relative absence of expression of NPY in phaeochromocytomas from VHL patients when compared with other groups appears to be largely independent of differences in catecholamine production and is consistent with a unique phenotype in VHL syndrome.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/química , Neuropéptido Y/análisis , Feocromocitoma/química , Enfermedad de von Hippel-Lindau/metabolismo , Adulto , Anciano , Análisis de Varianza , Femenino , Humanos , Inmunohistoquímica , Modelos Lineales , Masculino , Persona de Mediana Edad , Neoplasia Endocrina Múltiple Tipo 2a/química , Neuropéptido Y/genética , Feniletanolamina N-Metiltransferasa/análisis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Fluorescence imaging techniques for recording cytosolic [Ca(2+)](i) from single chromaffin cells were used to characterize and discriminate between cell subpopulations containing gamma-aminobutyric acid (GABA)(A) and GABA(B) receptor subtypes. By combining this methodology with the immunoidentification of individual chromaffin cells using specific antibodies against tyrosine hydroxylase (TH), phenyl-etanolamine-N-methyl transferase (PNMT), and glutamic acid decarboxylase (GAD) linked to different fluorescent probes, we have been able to ascribe single-cell calcium responses to identified adrenergic and noradrenergic chromaffin cells. GAD enzyme is present in 30% of the chromaffin cell population, located primarily in adrenergic cells; 86% of GAD(+) cells were also PNMT(+). GAD expression was not correlated with the presence of GABA receptors. GABA-responsive cells were found with equal frequency in the GAD(+) and GAD(-) groups. However, the expression of GABA receptors was correlated with the adrenergic phenotype. [Ca(2+)](i) responses to GABA were found more frequently in adrenergic than in noradrenergic cells. GABA(A) receptors are more evenly distributed; about 90% of GABA-responsive cells have them. GABA(B) receptors have a more restricted distribution (present in 45% of responding cells). The coexpression of both GABA(A) and GABA(B) subtypes is the rule; only a minor subpopulation (about 12%) displays exclusively GABA(B) receptors. GABA receptor subtypes are distributed in a similar way when chromaffin cells are separated according to GAD(+)/GAD(-) or PNMT(+)/PNMT(-) classifications, with only minor differences. These data indicate that the intrinsic GABAergic system in the adrenal medulla is not designed as a paracrine model in which a group of cells specializes in transmitter synthesis and a different group serves as a specific target.
Asunto(s)
Células Cromafines/química , Epinefrina/fisiología , Norepinefrina/fisiología , Receptores de GABA/metabolismo , Animales , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Epinefrina/biosíntesis , Glutamato Descarboxilasa/análisis , Glutamato Descarboxilasa/biosíntesis , Norepinefrina/biosíntesis , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/biosíntesis , Receptores de GABA/biosíntesis , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/biosíntesis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.
Asunto(s)
Bulbo Raquídeo/citología , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Receptores Adrenérgicos/fisiología , Estilbamidinas , Sistema Nervioso Simpático/citología , Animales , Colorantes Fluorescentes , Bulbo Raquídeo/química , Bulbo Raquídeo/enzimología , Microscopía Electrónica , Proteínas del Tejido Nervioso/inmunología , Vías Nerviosas , Neuronas/enzimología , Neuronas/ultraestructura , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/inmunología , Ratas , Médula Espinal/química , Médula Espinal/citología , Médula Espinal/enzimología , Sistema Nervioso Simpático/química , Sistema Nervioso Simpático/enzimología , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/inmunologíaRESUMEN
Agonists of the mu-opioid receptor (MOR) have profound effects on blood pressure, heart rate, and respiration that may be mediated by C1 adrenergic neurons in the rostral ventrolateral medulla (RVL). C1 neurons are sympathoexcitatory and are involved in both tonic and reflex regulation of sympathetic outflow. This study was designed to determine whether C1 neurons, or their afferents, contain MOR. C1 neurons were identified by using an antibody against the epinephrine synthesizing enzyme phenylethanolamine-N-methyl transferase (PNMT), whereas MOR was localized by using an antipeptide antibody that recognizes the cloned MOR, MOR1. Combined immunoperoxidase and immunogold methods were used to examine the cellular distribution of MOR1 relative to PNMT-containing neurons in the RVL. MOR1 was found in 22% of PNMT-containing dendrites (n = 392), whereas MOR1-containing axons or axon terminals contacted 14% of PNMT-containing dendrites. This distribution was heterogenous with regard to dendritic size: PNMT-labeled dendrites containing MOR1 were usually large (60% were >1.2 microm), whereas PNMT-containing dendrites that received MOR1-labeled afferents were usually small (79% were <1.2 microm). Individual dendrites rarely contained MOR1 at both pre- and postsynaptic sites. Together these results suggest that MOR agonists may directly influence the activity of C1 neurons, as well as the activity of select afferents to these cells. Plasmalemmal membrane labeling for MOR1 was more frequent in smaller PNMT-containing dendrites, suggesting that postsynaptic receptors are more readily available for ligand binding in small dendrites, although the receptor was more frequently detected in larger PNMT dendrites. The selective distribution of MORs to specific pre- and postsynaptic sites suggests the receptor may be selectively trafficked to positions where it may regulate afferent activity that is heterogeneously distributed along the dendritic tree of C1 neurons.
Asunto(s)
Bulbo Raquídeo/química , Bulbo Raquídeo/citología , Neuronas Aferentes/química , Receptores Opioides mu/análisis , Animales , Presión Sanguínea , Dendritas/química , Dendritas/enzimología , Dendritas/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Neuronas Aferentes/enzimología , Neuronas Aferentes/ultraestructura , Feniletanolamina N-Metiltransferasa/análisis , Ratas , Ratas Sprague-Dawley , Transactivadores/análisisRESUMEN
Adrenomedullin, originally identified in the adrenal medulla, has binding sites in the adrenal gland; however, its role in the adrenal medulla is unclear. This study was designed to characterise adrenomedullin binding sites in the rat adrenal medulla, using ligand binding studies, immunocytochemistry, and mRNA analysis. A single population of specific adrenomedullin receptors was identified in adrenal medullary homogenates. 125I-Adrenomedullin was displaced only by adrenomedullin1-50 and not by calcitonin gene-related peptide or amylin at concentrations up to 100 nmol/L. The receptor K(D) was 3.64 nmol/L with a receptor density of 570 fmol/mg of protein. Analysis of mRNA revealed that the genes encoding both the putative adrenomedullin receptors, termed calcitonin receptor-like receptor (CRLR) and L1, were expressed in the rat adrenal medulla. Dual-colour indirect-labelled immunofluorescence was used to localise phenylethanolamine N-methyltransferase (PNMT) and the adrenomedullin receptor in the same section. PNMT is the enzyme that converts noradrenaline to adrenaline and is not expressed in noradrenaline-secreting cells. These studies revealed that both CRLR and L1 were expressed only in cells that did not express PNMT, suggesting that adrenomedullin receptors are only found in noradrenaline-secreting cells. Further evidence to support this conclusion was provided by the demonstration of colocalisation of adrenomedullin receptors with dopamine beta-hydroxylase, confirming the presence of the receptors in medullary chromaffin cells. Taken together, these data suggest that adrenomedullin acts through a specific adrenomedullin receptor in the rat adrenal medulla. RT-PCR and northern blot analysis revealed greater abundance of mRNA for L1 than for CRLR, possibly suggesting that L1 may be the major adrenomedullin receptor expressed in this tissue. As it has been reported that adrenomedullin is synthesised predominantly by adrenaline-secreting cells, it appears likely that adrenomedullin is a paracrine regulator in the adrenal medulla.
Asunto(s)
Médula Suprarrenal/química , Proteínas de la Membrana/análisis , Norepinefrina/metabolismo , Receptores de Péptidos , Médula Suprarrenal/citología , Médula Suprarrenal/enzimología , Hormona Adrenocorticotrópica/metabolismo , Hormona Adrenocorticotrópica/farmacología , Adrenomedulina , Secuencia de Aminoácidos , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Células Cromafines/química , Células Cromafines/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Hemocianinas/genética , Radioisótopos de Yodo , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Feniletanolamina N-Metiltransferasa/análisis , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores de AdrenomedulinaRESUMEN
Adrenergic (C1) neurons in the rostral ventrolateral medulla (RVL) are sympathoexcitatory and project directly to sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord. C1 neurons also contain glutamate which may mediate the excitatory effects of RVL stimulation on SPNs through the N-methyl-D-aspartate (NMDA)-type receptor. Dual-labeling immunocytochemistry, combined with electron microscopy, was used to determine if NMDA receptors are located post-synaptic to adrenergic terminals in the spinal cord. Adrenergic terminals were labeled using an antibody directed against phenylethanolamine-N-methyl transferase (PNMT) and the NMDA receptor was identified using an antibody directed against the R1 subunit of the receptor (NMDAR1). NMDAR1 was found primarily in large and small dendrites and a few perikarya. The presence of NMDAR1 in the dendritic targets of PNMT-containing terminals was quantified in spinal cords sectioned either horizontally or coronally. PNMT-labeled terminals formed asymmetric synapses on dendrites containing immunogold labeling for NMDAR1, but NMDAR1 was more often detected in the targets of PNMT terminals when spinal cords were sectioned horizontally (59%) rather than coronally (28%). This difference in prevalence of NMDAR1 in targets of PNMT terminals is likely due to the preferential orientation of SPN dendrites in the horizontal plane, since longer dendritic shafts were visible in horizontal sections. When NMDAR1 was present in the dendritic targets of many adrenergic terminals, it was usually located at sites distal to the adrenergic input. We conclude that NMDA receptor ligands are likely to modulate the activity of dendritic targets of adrenergic terminals in the spinal cord, but are not closely associated with adrenergic synaptic input.
Asunto(s)
Dendritas/ultraestructura , Neuronas/ultraestructura , Receptores de N-Metil-D-Aspartato/análisis , Médula Espinal/ultraestructura , Sinapsis/ultraestructura , Animales , Axones/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Terminaciones Nerviosas/ultraestructura , Feniletanolamina N-Metiltransferasa/análisis , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
The distribution and ascending projections to the hypothalamic paraventricular nucleus of phenylethanolamine N-methyltransferase (PNMT)-immunoreactive perikaria were studied in adult pigeons using a combination of retrograde transport of Fluorogold injected into the paraventricular nucleus, and double immunohistochemical procedures for PNMT, tyrosine hydroxylase and neuropeptide Y. PNMT-immunoreactive cell bodies were found in the subtrigeminal reticular nucleus of the ventrolateral medulla and in the nucleus of the solitary tract, mainly in the subnuclei: medialis superficialis, pars posterior, and medialis ventralis, pars posterior. PNMT-immunoreactive perikaria were also tyrosine hydroxylase immunoreactive, and are located within the rostral tyrosine hydroxylase immunoreactive cell groups of these areas. No perikaria double-labeled for neuropeptide Y and PNMT were found. Retrograde labeled cell bodies were observed in the subtrigeminal reticular nucleus and in the nucleus of the solitary tract. PNMT-immunoreactive retrogradely labeled cells were mainly observed in the subtrigeminal reticular nucleus. These data suggest the presence in the pigeon of medullary adrenergic cell groups partially comparable to mammalian C1 and C2 groups. Comparison of these results with data previously obtained in amphibians and reptiles suggests that the presence of a hypothalamically-projecting C1-like group might be a plesiomorphic medullary attribute in amniotes, whereas the variable presence of C2 and C3-like groups, as well as the content of NPY in the putative adrenergic perikaria, seem to be species-specific.
Asunto(s)
Mapeo Encefálico , Columbidae , Bulbo Raquídeo/anatomía & histología , Neuronas/química , Núcleo Hipotalámico Paraventricular/anatomía & histología , Feniletanolamina N-Metiltransferasa/análisis , Animales , Fluoroinmunoensayo , Inmunohistoquímica , Bulbo Raquídeo/química , Bulbo Raquídeo/ultraestructura , Vías Nerviosas , Neuronas/inmunología , Neuropéptido Y/análisis , Núcleo Hipotalámico Paraventricular/química , Núcleo Hipotalámico Paraventricular/ultraestructura , Feniletanolamina N-Metiltransferasa/inmunología , Especificidad de la Especie , Tirosina 3-Monooxigenasa/análisisRESUMEN
BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.
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Médula Suprarrenal/química , Médula Suprarrenal/citología , Células Cromafines/química , Citometría de Flujo/métodos , Médula Suprarrenal/enzimología , Animales , Bovinos , Separación Celular , Células Cromafines/enzimología , Dopamina beta-Hidroxilasa/análisis , Dopamina beta-Hidroxilasa/metabolismo , Epinefrina/análisis , Inmunohistoquímica , Norepinefrina/análisis , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/metabolismo , Factores de TiempoRESUMEN
In patients with spinal cord injury, cardiovascular disturbances such as hypotension, bradycardia and autonomic dysreflexia can be directly linked to abnormalities of central autonomic control. To date, the changes in bulbospinal innervation of sympathetic preganglionic neurons after compressive spinal cord injury have not been investigated. Thus, we examined the effect of varying severity of compressive spinal cord injury on neurons of the rostro-ventro-lateral medulla, a nucleus of key importance in cardiovascular control. Adult rats with 20 g, 35 g and 50 g clip compression injuries (n= 18) of the cord at T1 and uninjured controls (n=13) were studied. Neurons in the rostro-ventro-lateral medulla with preserved spinal connections eight weeks after spinal cord injury were identified by retrograde labelling with 4% FluoroGold introduced into the cord at T6. Bulbospinal neurons in the rostro-ventro-lateral medulla were also examined immunocytochemically for the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase. In control rats an average of 451+/-12 rostro-ventrolateral medulla neurons were phenylethanolamine-N-methyltransferase positive. Of these, 213+/-6 projected to the T6 spinal cord. The number of rostro-ventro-lateral medulla neurons retrogradely labelled by FluoroGold decreased as a linear function of severity of spinal cord injury (r= -0.95; P<0.0001). After 50g spinal cord injury at T1, only 7+/-1 rostro-ventro-lateral medulla neurons were labelled by FluoroGold, of which 6+/-1 were phenylethanolamine-N-methyltransferase positive. Moreover, the number of phenylethanolamine-N-methyltransferase positive rostro-ventro-lateral medulla neurons decreased to 361+/-16 after 50 g spinal cord injury. We conclude that compressive spinal cord injury results in disconnection of rostro-ventro-lateral medulla neurons, which project to the thoracic spinal cord, and that these changes vary with the severity of injury. The majority of these axotomized rostro-ventro-lateral medulla neurons maintain their immunopositivity for the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase.
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Bulbo Raquídeo/fisiopatología , Neuronas/fisiología , Compresión de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Estilbamidinas , Animales , Transporte Axonal , Encéfalo/enzimología , Encéfalo/patología , Encéfalo/fisiopatología , Femenino , Colorantes Fluorescentes , Laminectomía , Masculino , Bulbo Raquídeo/enzimología , Bulbo Raquídeo/fisiología , Feniletanolamina N-Metiltransferasa/análisis , Ratas , Ratas Wistar , Valores de Referencia , Médula Espinal/fisiología , Compresión de la Médula Espinal/enzimología , Compresión de la Médula Espinal/patologíaRESUMEN
In the paracervical ganglion (PCG) of the rat, double-labelling immunofluorescence for catecholamine-synthesizing enzymes and HPLC measurement of catecholamine contents were first performed to evaluate whether intraganglionic small intensely fluorescent (SIF) cells are capable of synthesizing adrenaline. Immunolabelling for tyrosine hydroxylase (TH), dopamine beta-hydroxylase and phenylethanolamine-N-methyl transferase (PNMT) occurred in all SIF cells of the PCG, thus demonstrating the presence of all the enzymes required for adrenaline biosynthesis. Adrenaline levels were undetectable in the PCG but to test the hypothesis that PNMT is active in SIF cells, catecholamines were measured in ganglia of rats pretreated with pargyline, an inhibitor of the monoamine oxidase, the major enzyme involved in the catecholamine degradation. Pargyline treatment increased adrenaline levels in the PCG, thus demonstrating that SIF cells are capable of adrenaline synthesis. The undetectable levels of adrenaline in the PCG of untreated rats suggested a slow rate of biosynthesis of adrenaline in the ganglion. Furthermore, the use of double-labelling showed that SIF cells of the PCG were stained for muscarinic receptors and were approached by varicose ChAT-immunoreactive nerve fibres. Nerve fibres immunoreactive for ChAT were also observed associated with nerve cell bodies of ganglion neurones. Following deafferentation of the PCG, the ChAT-immunoreactive nerve fibres surrounding nerve cell bodies totally disappeared indicating their preganglionic origin, while those associated with SIF cells did not degenerate, which demonstrate that they derived from intraganglionic cholinergic neurones. Taken together, the results show that adrenaline may be a transmitter for SIF cells in the PCG and suggest that cholinergic neurones of the parasympathetic division of the PCG can modulate the SIF cell activity through the activation of muscarinic receptors.
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Fibras Autónomas Posganglionares/enzimología , Ganglios Simpáticos/enzimología , Neuronas/química , Receptores Muscarínicos/análisis , Vías Aferentes/fisiología , Animales , Fibras Autónomas Posganglionares/citología , Línea Celular , Tamaño de la Célula , Colina O-Acetiltransferasa/análisis , Dopamina beta-Hidroxilasa/análisis , Epinefrina/biosíntesis , Femenino , Técnica del Anticuerpo Fluorescente , Ganglios Simpáticos/citología , Inmunohistoquímica , Feniletanolamina N-Metiltransferasa/análisis , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/análisisRESUMEN
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.
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Catecolaminas/biosíntesis , Ganglios Autónomos/enzimología , Corazón/inervación , Histamina/biosíntesis , Acetilcolina/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales , Catecolaminas/análisis , Catecolaminas/inmunología , Reacciones Cruzadas , Dopa-Decarboxilasa/análisis , Dopa-Decarboxilasa/inmunología , Dopa-Decarboxilasa/metabolismo , Dopamina/análisis , Dopamina/biosíntesis , Dopamina/inmunología , Dopamina beta-Hidroxilasa/análisis , Dopamina beta-Hidroxilasa/inmunología , Dopamina beta-Hidroxilasa/metabolismo , Femenino , Ganglios Autónomos/química , Ganglios Autónomos/citología , Histamina/análisis , Histamina/inmunología , Histidina Descarboxilasa/análisis , Histidina Descarboxilasa/inmunología , Histidina Descarboxilasa/metabolismo , Humanos , Indoles/metabolismo , Masculino , Persona de Mediana Edad , Neuronas/química , Neuronas/enzimología , Norepinefrina/análisis , Norepinefrina/biosíntesis , Norepinefrina/inmunología , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/inmunología , Feniletanolamina N-Metiltransferasa/metabolismo , Serotonina/análisis , Serotonina/biosíntesis , Serotonina/inmunología , Triptófano Hidroxilasa/análisis , Triptófano Hidroxilasa/inmunología , Triptófano Hidroxilasa/metabolismo , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/inmunología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase-antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.
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Fibras Adrenérgicas/ultraestructura , Catecolaminas , Fibras Colinérgicas/ultraestructura , Globo Pálido/anatomía & histología , Neuronas Aferentes/ultraestructura , Acetilcolinesterasa/análisis , Fibras Adrenérgicas/química , Animales , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/química , Dopamina beta-Hidroxilasa/análisis , Globo Pálido/química , Técnicas para Inmunoenzimas , Masculino , Microscopía Electrónica , Neuronas Aferentes/química , Feniletanolamina N-Metiltransferasa/análisis , Ratas , Ratas Wistar , Tirosina 3-Monooxigenasa/análisisRESUMEN
Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.
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Canales de Calcio Tipo N , Canales de Calcio/fisiología , Células Cromafines/fisiología , Adenosina Trifosfato/farmacología , Neoplasias de las Glándulas Suprarrenales/patología , Glándulas Suprarrenales/citología , Adulto , Animales , Bario/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Bovinos , Recuento de Células , Separación Celular , Células Cultivadas , Células Cromafines/citología , Quistes , Dopamina beta-Hidroxilasa/análisis , Encefalina Metionina/farmacología , Femenino , Humanos , Inmunohistoquímica , Feniletanolamina N-Metiltransferasa/análisisRESUMEN
Phylogenetic, physiological and morphological evidence indicates that interactions between chromaffin and adrenocortical cells are involved in the differentiation and maintenance of function of both cell types. Chromaffin-adrenocortical interaction has become recognized as an important component of adrenocortical regulation; however, the mechanisms by which chromaffin cells modulate adrenocortical function are not well understood. To study directly chromaffin-adrenocortical cellular interactions, we developed primary frog (Rana pipiens) adrenal co-cultures. In these co-cultures, chromaffin cells extend processes that project towards or onto adrenocortical cells, mimicking their organization in vivo and indicating a potential for interaction between the two cell types. Cell survival and differentiation were optimized using a combination of NGF, FGF and histamine to enhance neurite outgrowth and fetal calf serum plus 10(-10) M ACTH to maintain steroidogenesis. Characterization of the cells by immunocytochemistry and histochemistry showed that chromaffin cells maintain expression of catecholamine biosynthetic enzymes and that adrenocortical cells maintain expression of steroidogenic enzymes. Furthermore, chromaffin cells release catecholamines upon stimulation with carbamylcholine or potassium while adrenocortical cells sustain a basal secretion rate of aldosterone and corticosterone that is augmented 10-40-fold by 0.1 nM to 10 nM ACTH. We therefore propose that these co-cultures serve as a useful model system to study the cellular and molecular mechanisms by which chromaffin cells modulate adrenocortical cell function.