RESUMO
Neuroendocrine tumours (NETs) are rare, heterogeneous, and often hormonally active neoplasms. Nuclear medicine (NM) imaging using single photon- and positron-emitting radiopharmaceuticals allows sensitive and highly specific molecular imaging of NETs, complementary to anatomy-based techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI). Somatostatin-receptor scintigraphy is a whole-body imaging technique widely used for diagnosis, staging and restaging of NETs. The increasing availability of hybrid single-photon emission CT (SPECT)/CT cameras now offers superior accuracy for localization and functional characterization of NETs compared to traditional planar and SPECT imaging. The potential role of positron-emission tomography (PET) tracers in the functional imaging of NETs is also being increasingly recognized. In addition to 2-[(18)F]-fluoro-2-deoxy-d-glucose (FDG), newer positron-emitting radiopharmaceuticals such as (18)F-dihydroxyphenylalanine (DOPA) and (68)Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) peptides, show promise for the future. This article will summarize the role of current and emerging radiopharmaceuticals in NM imaging of this rare but important group of tumours.
Assuntos
Tumores Neuroendócrinos/diagnóstico por imagem , Sistema Cromafim/diagnóstico por imagem , Di-Hidroxifenilalanina/análogos & derivados , Fluordesoxiglucose F18 , Radioisótopos de Gálio , Humanos , Tumores Neuroendócrinos/patologia , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.
Assuntos
Neurossecreção/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Sistema Cromafim/fisiologia , Humanos , Neurotransmissores/metabolismo , Neurotransmissores/fisiologia , Células PC12 , Ratos , Transmissão Sináptica/fisiologiaRESUMO
BACKGROUND: Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. METHODS: fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. RESULTS: fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. CONCLUSIONS: Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.
Assuntos
Doença Celíaca/genética , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Chaperonas Moleculares/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Doença Celíaca/metabolismo , Linhagem Celular , Criança , Sistema Cromafim/metabolismo , Enterócitos/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fases de Leitura Aberta , RNA Mensageiro/análise , Valores de Referência , Adulto JovemRESUMO
The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.
Assuntos
Cálcio/fisiologia , Catecolaminas/metabolismo , Sistema Cromafim/metabolismo , Ionóforos/farmacologia , Sódio/fisiologia , Animais , Bovinos , Células Cultivadas , Sistema Cromafim/ultraestrutura , Microscopia Eletrônica , Monensin/farmacologia , Fatores de TempoRESUMO
High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.
Assuntos
Proteínas de Membrana/análise , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/análise , Neurônios/análise , Feocromocitoma/análise , Animais , Axônios/análise , Bucladesina/farmacologia , Diferenciação Celular , Linhagem Celular , Membrana Celular/análise , Sistema Cromafim/análise , Sistema Cromafim/citologia , Sistema Cromafim/ultraestrutura , Proteína GAP-43 , Complexo de Golgi/análise , Imuno-Histoquímica , Lisossomos/análise , Microscopia Eletrônica , Microvilosidades/análise , Neurônios/ultraestrutura , Feocromocitoma/ultraestrutura , Pseudópodes/análiseRESUMO
The authors of this study examine the question of whether the so-called enterochromaffin or argentaffin cells of the gastrointestinal tract should be considered as a single cell type. The systematic application of purely morphologic methods has led to the conclusion that the epithelium of the gastrointestinal mucosa comprises endocrine cells of several types. This conclusion is primarily based on the uneven and characteristic distribution of the various cell types along the intestinal tract, an observation precluding the interpretation that the different types correspond to diverse functional stages of the same cell. A specific endocrine function may be attributed to each of the given cell types recognized so far on account of their appearance and their localization in characteristic areas of the gastrointestinal tract. It is acknowledged, however, that a purely morphological study leaves room for doubt. The first cell type is probably responsible for the formation of 5-hydroxytryptamine. Cells of type II are morphologically comparable to the pancreatic A cells and may, therefore, be called intestinal A cells. Cell type III comprises intestinal D cells since their appearance corresponds to that of pancreatic D cells. Cell type IV might well be responsible for catecholamine production, whereas gastrin is in all probability produced in endocrine cell type V. As yet, the thorough morphological study of the gastrointestinal epithelium does not provide information as to additional distinct cellular sites of production of the several other hormones isolated from different parts of the gut.
Assuntos
Sistema Cromafim/citologia , Mucosa Gástrica/citologia , Mucosa Intestinal/citologia , Animais , Grânulos Citoplasmáticos , Células Epiteliais , Feminino , Masculino , Microscopia Eletrônica , Ratos , Ribossomos , Serotonina/biossínteseRESUMO
The effect of 0.5-1.0 microM taxol, a potent promoter of microtubule polymerization in vitro, was studied on the secretory activity of chromaffin cells of the adrenal medulla. Taxol was found to have a dual effect: the long-term effect (after a 1-h incubation) of taxol was to induce almost complete inhibition of catecholamine release, whereas after a short incubation (10 min) a massive, nicotine-independent release of catecholamine was produced. From results obtained using the patch-clamp technique to study the Ca++-dependent K+ channels (Ic channels), it was possible to conclude that taxol probably provokes an augmentation of free [Ca++]i in the cytoplasm, values increasing from 10(-8) M at rest to several 10(-7) M. The increased spontaneous release of stored neurohormones and the increased frequency of opening of Ic channels occur simultaneously and could both originate from a rise of [Ca++]i upon taxol addition. Immunofluorescence and ultrastructural studies showed that 13-h taxol treatment of chromaffin cells led to a different distribution of secretory organelles, and also to microtubule reorganization. In treated cells, microtubules were found to form bundles beneath the cell membrane and, at the ultrastructural level, to be packed along the cell axis. It is concluded that in addition to its action on microtubules, the antitumor drug taxol has side effects on the cell secretory activity, one of them being to modify free [Ca++]i.
Assuntos
Medula Suprarrenal/citologia , Alcaloides/farmacologia , Sistema Cromafim/fisiologia , Animais , Antineoplásicos Fitogênicos , Cálcio/metabolismo , Cálcio/farmacologia , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Sistema Cromafim/efeitos dos fármacos , Sistema Cromafim/ultraestrutura , Dimetil Sulfóxido/farmacologia , Eletrofisiologia , Imunofluorescência , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Paclitaxel , Potássio/metabolismoRESUMO
The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 X 10(4) cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (alpha-amanitin) and processing (9-beta-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the density-mediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.
Assuntos
Comunicação Celular , Sistema Cromafim/enzimologia , Células Enterocromafins/enzimologia , Tirosina 3-Mono-Oxigenase/biossíntese , Acetilcolinesterase/biossíntese , Animais , Bovinos , Diferenciação Celular , Células Cultivadas , Células Enterocromafins/citologia , Regulação da Expressão Gênica , L-Lactato Desidrogenase/biossíntese , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.
Assuntos
Medula Suprarrenal/metabolismo , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Exocitose , Animais , Cálcio/metabolismo , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Dopamina beta-Hidroxilase/metabolismo , Soluções Hipertônicas , Pressão OsmóticaRESUMO
We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.
Assuntos
Calmodulina/farmacologia , Proteínas de Transporte/farmacologia , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Proteínas de Membrana/farmacologia , Proteínas/farmacologia , Tirosina 3-Mono-Oxigenase , Proteínas de Transporte Vesicular , Proteínas 14-3-3 , Animais , ATPase de Ca(2+) e Mg(2+)/fisiologia , Bovinos , Permeabilidade da Membrana Celular/fisiologia , Sistema Cromafim/citologia , Sistema Cromafim/metabolismo , Citosol/metabolismo , Digitonina , Exocitose/fisiologia , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/biossíntese , Ratos , Ratos Wistar , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Glândulas Suprarrenais/citologia , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Variação Antigênica , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Centrifugação com Gradiente de Concentração , Grânulos Cromafim/ultraestrutura , Cromatografia em Gel , Meia-Vida , Receptores Nicotínicos/imunologiaRESUMO
Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that mediate release of catecholamines from the cells in response to synaptic input, and resemble neuronal AChRs in pharmacology and antigenic profile. Results presented here show that a cAMP-dependent process enhances the function of adrenal chromaffin AChRs as a population in the plasma membrane. This was demonstrated by showing that cAMP analogues cause specific increases both in the level of nicotine-induced catecholamine release from the cells and in the level of the nicotine-induced conductance change occurring in the cells. Neither de novo synthesis of receptors nor transport of preexisting intracellular receptors to the plasma membrane is necessary for the enhancement. The responsiveness of AChRs to regulation by the cAMP-dependent process appears to depend on the length of time the receptors have been on the cell surface. AChRs newly inserted into the plasma membrane generate a greater nicotinic response than do older AChRs and, unlike older AChRs, their response to agonist is not enhanced after treatment of the cells with cAMP analogues. The findings indicate that the AChRs and/or associated components undergo a maturation in the plasma membrane that alters their function and their regulation by secondary messenger systems.
Assuntos
Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , AMP Cíclico/farmacologia , Receptores Nicotínicos/metabolismo , Glândulas Suprarrenais/citologia , Animais , Catecolaminas/metabolismo , Bovinos , Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , Eletrofisiologia , Nicotina/farmacologia , Receptores Nicotínicos/efeitos dos fármacosRESUMO
A nucleus of catecholamine-containing cells bordering the preoptic recess of the toad hypothalamus has been studied by both fluorescence histochemical and electron microscopic methods. The perikarya of these cells form one to three rows immediately subjacent to the ependyma. They send brightly fluorescent apical processes between the ependymal cells to the ventricular surface, and also give rise to long basal processes, the proximal portions of which are also fluorescent. These cells contain two distinctive constitutents: juxtanuclear bundles of tightly packed filaments, the members of which are separated from one another by only approximately 100 A, and large numbers of dense-cored vesicles (400-2200 A in diameter), which appear to arise from an agranular tubular reticulum distinct from the Golgi apparatus. Axons containing either clear vesicles alone or clear and dense-cored vesicles form synapses on the subependymal cells, but no evidence has been found that the subependymal cells themselves form presynaptic contacts, or that axons originate from them. The cytological characteristics of these catecholamine-containing cells, plus the fact that they border directly on the cerebrospinal fluid, suggest that they may be more closely related to peripheral chromaffin cells than to the other cell types intrinsic to the central nervous system, and the name "encephalo-chromaffin cells" is therefore proposed for them. The possible functions of such cells in the central nervous system are discussed.
Assuntos
Anuros/anatomia & histologia , Catecolaminas/análise , Hipotálamo/citologia , Animais , Axônios/química , Líquido Cefalorraquidiano/citologia , Sistema Cromafim/citologia , Grânulos Citoplasmáticos , Retículo Endoplasmático , Epêndima/citologia , Feminino , Liofilização , Histocitoquímica , Hipotálamo/química , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Fibras Nervosas Mielinizadas/ultraestrutura , Coloração e Rotulagem , SinapsesRESUMO
The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues.
Assuntos
Membrana Celular/fisiologia , Grânulos Cromafim/fisiologia , Sistema Cromafim/fisiologia , Endocitose , Exocitose , Membranas Intracelulares/fisiologia , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Medula Suprarrenal/fisiologia , Animais , Antígenos de Superfície/análise , Bovinos , Dopamina beta-Hidroxilase/metabolismo , Imunofluorescência , Glicoproteínas/metabolismo , CinéticaRESUMO
The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.
Assuntos
Medula Suprarrenal/metabolismo , Membrana Celular/metabolismo , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Endocitose , Exocitose , Glicoproteínas/metabolismo , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Animais , Antígenos de Superfície/análise , Bovinos , Invaginações Revestidas da Membrana Celular/metabolismo , Glicoproteínas/imunologia , Ouro , Técnicas Imunológicas , Lisossomos/metabolismo , Proteínas de Membrana/imunologia , Microscopia EletrônicaRESUMO
The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Medula Suprarrenal/metabolismo , Cálcio/metabolismo , Comunicação Celular , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Exocitose , Trifosfato de Adenosina/farmacologia , Medula Suprarrenal/citologia , Medula Suprarrenal/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Benzofuranos , Células Cultivadas , Grânulos Cromafim/ultraestrutura , Citosol/metabolismo , Corantes Fluorescentes , Fura-2 , Camundongos , Nicotina/farmacologiaRESUMO
When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.
Assuntos
Grânulos Cromafim/efeitos dos fármacos , Sistema Cromafim/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Fusão de Membrana/efeitos dos fármacos , Proteínas/farmacologia , Animais , Anexina A7 , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Bovinos , Epinefrina/metabolismo , Exocitose/efeitos dos fármacos , Microscopia Eletrônica , Polilisina/farmacologia , Relação Estrutura-AtividadeRESUMO
Immunoreactive serotonin was detected in the lumen of the proximal jejunum of food-deprived cats. During perfusion of this intestinal segment in vivo, there was a constant basal rate of intraluminal secretion of this amine. The rate of secretion was significantly increased during efferent electrical stimulation of the cut cervical vagal nerves. This stimulatory effect was not altered after bilateral adrenalectomy was performed in the same animals. A synchronous release of substance P into the gut lumen was also demonstrated during vagal stimulation. During the period of increased intraluminal secretion of immunoreactive serotonin, there was no demonstrable change in the portal or systemic blood levels of this amine.
Assuntos
Sistema Cromafim/metabolismo , Células Enterocromafins/metabolismo , Jejuno/metabolismo , Serotonina/metabolismo , Nervo Vago/fisiologia , Adrenalectomia , Animais , Gatos , Estimulação Elétrica , Radioimunoensaio , Substância P/metabolismoRESUMO
A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.
Assuntos
Medula Suprarrenal/análise , Grânulos Cromafim/análise , Sistema Cromafim/análise , Endorfinas/biossíntese , Encefalinas/biossíntese , Precursores de Proteínas/análise , Proteínas/análise , Animais , Bovinos , Encefalina Leucina , Encefalina Metionina , Peso Molecular , Oligopeptídeos/análise , Fragmentos de Peptídeos/análise , TripsinaRESUMO
Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.