RESUMEN
Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.
Asunto(s)
Gránulos Cromafines/fisiología , Receptores Sensibles al Calcio/fisiología , Sinaptotagminas/genética , Sinaptotagminas/fisiología , Acetilcolina/farmacología , Animales , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Fenómenos Electrofisiológicos , Exocitosis , Femenino , Cinética , Masculino , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células PC12 , Ratas , Proteínas SNARE/metabolismo , Fracciones Subcelulares/metabolismo , Sinaptotagmina I/fisiologíaRESUMEN
Many of the molecular players in the stimulus-secretion chain are similarly active in neurosecretion and catecholamine release. Therefore, studying chromaffin cells uncovered many details of the processes of docking, priming, and exocytosis of vesicles. However, morphological specializations at synapses, called active zones (AZs), confer extra speed of response and another layer of control to the fast release of vesicles by action potentials. Work at the Calyx of Held, a glutamatergic nerve terminal, has shown that in addition to such rapidly released vesicles, there is a pool of "Slow Vesicles," which are held to be perfectly release-competent, but lack a final step of tight interaction with the AZ. It is argued here that such "Slow Vesicles" have many properties in common with chromaffin granules. The added complexity in the AZ-dependent regulation of "Fast Vesicles" can lead to misinterpretation of data on neurosecretion. Therefore, the study of Slow Vesicles and of chromaffin granules may provide a clearer picture of the early steps in the highly regulated process of neurosecretion.
Asunto(s)
Gránulos Cromafines/fisiología , Neurosecreción , Animales , Gránulos Cromafines/metabolismo , Humanos , Transmisión SinápticaRESUMEN
Large dense core vesicles and chromaffin granules accumulate solutes at large concentrations (for instance, catecholamines, 0.5-1 M; ATP, 120-300 mM; or Ca2+, 40 mM (12)). Solutes seem to aggregate to a condensed protein matrix, which is mainly composed of chromogranins, to elude osmotic lysis. This association is also responsible for the delayed release of catecholamines during exocytosis. Here, we compile experimental evidence, obtained since the inception of single-cell amperometry, demonstrating how the alteration of intravesicular composition promotes changes in the quantum characteristics of exocytosis. As chromaffin cells are large and their vesicles contain a high concentration of electrochemically detectable species, most experimental data comes from this cell model.
Asunto(s)
Catecolaminas/metabolismo , Gránulos Cromafines/metabolismo , Exocitosis , Animales , Gránulos Cromafines/fisiología , Cromograninas/metabolismo , Técnicas Electroquímicas/métodos , Humanos , Transmisión SinápticaRESUMEN
Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells.
Asunto(s)
Gránulos Cromafines/metabolismo , Técnicas Electroquímicas/métodos , Exocitosis , Potenciales de Acción , Animales , Gránulos Cromafines/fisiología , Citofotometría/instrumentación , Citofotometría/métodos , Técnicas Electroquímicas/instrumentación , HumanosRESUMEN
The accumulation of neurotransmitters within secretory vesicles (SVs) far exceeds the theoretical tonic concentrations in the cytosol, a phenomenon that has captivated the attention of scientists for decades. For instance, chromaffin granules can accumulate close to molar concentrations of catecholamines, along with many other products like ATP, calcium, peptides, chromogranins, ascorbate, and other nucleotides. In this short review, we will summarize the interactions that are currently believed to occur between the elements that make up the vesicular cocktail in the acidic environment of SVs, and how they permit the accumulation of such high concentrations of certain components. In addition, we will examine how the vesicular cocktail regulates the exocytosis of neurotransmitters. In this review, we have highlighted the mechanisms that permit the storage of neurotransmitters and hormones inside secretory vesicles. We also have proposed a novel model based in the intravesicular interactions of the main components of this inner cocktail - catecholamines, ATP, and chromogranins - to allow the accumulation of near molar concentrations of transmitters in secretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).
Asunto(s)
Exocitosis/fisiología , Neurotransmisores/metabolismo , Vesículas Secretoras/fisiología , Adenosina Trifosfato/metabolismo , Animales , Gránulos Cromafines/fisiología , Cromograninas/metabolismo , Humanos , Modelos BiológicosRESUMEN
Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ~100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ~200-250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca(2+) ([Ca(2+)](i)) to ~0.5 µmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca(2+)](i) triggers a form of rapid "kiss-and-run" exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).
Asunto(s)
Glándulas Suprarrenales/fisiología , Catecolaminas/metabolismo , Células Cromafines/fisiología , Gránulos Cromafines/fisiología , Exocitosis/fisiología , Glándulas Suprarrenales/metabolismo , Animales , Calcio/metabolismo , Células Cromafines/metabolismo , Gránulos Cromafines/metabolismo , Cinética , Masculino , Concentración Osmolar , Ratas , Ratas Sprague-DawleyRESUMEN
We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.
Asunto(s)
Calcio/fisiología , Gránulos Cromafines/fisiología , Exocitosis , Animales , Bovinos , Citoplasma/fisiología , Dopamina beta-Hidroxilasa/metabolismo , Técnica del Anticuerpo Fluorescente , Inositol 1,4,5-Trifosfato/farmacología , Ionomicina/farmacología , Microinyecciones , Norepinefrina/metabolismo , Oocitos , Xenopus laevisRESUMEN
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.
Asunto(s)
Membrana Celular/fisiología , Gránulos Cromafines/fisiología , Sistema Cromafín/fisiología , Endocitosis , Exocitosis , Membranas Intracelulares/fisiología , Glicoproteínas de Membrana , Proteínas de la Membrana/metabolismo , Médula Suprarrenal/fisiología , Animales , Antígenos de Superficie/análisis , Bovinos , Dopamina beta-Hidroxilasa/metabolismo , Técnica del Anticuerpo Fluorescente , Glicoproteínas/metabolismo , CinéticaRESUMEN
Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody-antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell.
Asunto(s)
Médula Suprarrenal/fisiología , Endocitosis , Exocitosis , Médula Suprarrenal/efectos de los fármacos , Médula Suprarrenal/metabolismo , Animales , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Gránulos Cromafines/fisiología , Grupo Citocromo b/metabolismo , Dopamina beta-Hidroxilasa/metabolismo , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Sueros Inmunes , Cinética , Nicotina/farmacología , Veratridina/farmacologíaRESUMEN
Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.
Asunto(s)
Actinas/metabolismo , Corteza Suprarrenal/fisiología , Gránulos Cromafines/ultraestructura , Exocitosis , Actinas/efectos de los fármacos , Actinas/ultraestructura , Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/ultraestructura , Animales , Bovinos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Células Cultivadas , Gránulos Cromafines/efectos de los fármacos , Gránulos Cromafines/fisiología , Electrofisiología/métodos , Epinefrina/metabolismo , Cinética , Potenciales de la Membrana , Microscopía Electrónica , Nicotina/farmacología , Norepinefrina/metabolismo , Potasio/farmacología , Acetato de Tetradecanoilforbol/farmacología , Grabación en VideoRESUMEN
Chromogranin A (CHGA) and its derived peptides, which are stored and released from dense-core secretory granules of neuroendocrine cells, have been implicated as playing multiple roles in the endocrine, cardiovascular, and nervous systems. In this issue of the JCI, Mahapatra et al. present in vivo evidence for 2 important functions of CHGA: the regulation of catecholamine-containing dense-core chromaffin granule biogenesis in the adrenal gland and the control of blood pressure. Obliteration of CHGA expression in a KO mouse model led to decreased size and number of chromaffin granules as well as hypertension in these animals. Transgenic expression of human Chga and exogenous injection of human catestatin, a CHGA-derived nicotinic cholinergic antagonist, restored normal blood pressure in these mice. These results suggest a coupled relationship between CHGA-mediated chromaffin granule biogenesis, necessary for catecholamine storage, and catestatin-induced inhibition of cholinergic-stimulated catecholamine release, which regulates autonomic control of blood pressure.
Asunto(s)
Gránulos Cromafines/fisiología , Cromograninas/fisiología , Hipertensión/etiología , Animales , Presión Sanguínea/efectos de los fármacos , Catecolaminas/fisiología , Cromogranina A , Cromograninas/genética , Cromograninas/farmacología , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Fragmentos de Péptidos/farmacologíaRESUMEN
The secretory prohormone chromogranin A (CHGA) is overexpressed in essential hypertension, a complex trait with genetic predisposition, while its catecholamine release-inhibitory fragment catestatin is diminished, and low catestatin predicts augmented adrenergic pressor responses. These findings from studies on humans suggest a mechanism whereby diminished catestatin might increase the risk for hypertension. We generated Chga and humanized mice through transgenic insertion of a human CHGA haplotype in order to probe CHGA and catestatin in vivo. Chga mice displayed extreme phenotypic changes, including: (a) decreased chromaffin granule size and number; (b) elevated BP; (c) loss of diurnal BP variation; (d) increased left ventricular mass and cavity dimensions; (e) decreased adrenal catecholamine, neuropeptide Y (Npy), and ATP contents; (f) increased catecholamine/ATP ratio in the chromaffin granule; and (g) increased plasma catecholamine and Npy levels. Rescue of elevated BP to normalcy was achieved by either exogenous catestatin replacement or humanization of Chga mice. Loss of the physiological "brake" catestatin in Chga mice coupled with dysregulation of transmitter storage and release may act in concert to alter autonomic control of the circulation in vivo, eventuating in hypertension.
Asunto(s)
Cromograninas/genética , Cromograninas/fisiología , Hipertensión/etiología , Hipertensión/genética , Animales , Presión Sanguínea/fisiología , Gránulos Cromafines/patología , Gránulos Cromafines/fisiología , Cromogranina A , Ritmo Circadiano , Corticosterona/sangre , Epinefrina/sangre , Marcación de Gen , Humanos , Hipertensión/patología , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica , Neuropéptido Y/sangre , Norepinefrina/sangre , Renina/sangreRESUMEN
Dense vesicles can be observed in live bovine chromaffin cells using fluorescent reflection confocal microscopy. These vesicles display a similar distribution, cytoplasmic density and average size as the chromaffin granules visualized by electron microscopy. In addition, the acidic vesicles labeled with Lysotracker Red comprised a subpopulation of the vesicles that are visualized by reflection fluorescence. A combination of fluorescence reflection and transmitted light images permitted the movements of vesicles in relation to the cortical cytoskeleton to be studied. The movement of vesicles located on the outside of this structure was restricted, with an apparent diffusion coefficient of 1.0+/-0.4 x 10(-4) microm(2)/s. In contrast, vesicles located in the interior moved much more freely and escaped from the visual confocal plane. Lysotracker labeling was more appropriate to study the movement of the faster moving vesicles, whose diffusion coefficient was five times higher. Using this type of labeling we confirmed the restriction on cortical movement and showed a clear relationship between vesicle mobility and the kinetics of cytoskeletal movement on both sides of the cortical cytoskeleton. This relationship was further emphasized by studying cytoskeletal organization and kinetics. Indeed, an estimate of the size of the cytoskeletal polygonal cages present in the cortical region and in the cell interior agreed well with the calculation of the theoretical radius of the cages imprisoning vesicle movement. Therefore, these data suggest that the structure and kinetics of the cytoskeleton governs vesicle movements in different regions of chromaffin cells.
Asunto(s)
Actinas/metabolismo , Células Cromafines/fisiología , Gránulos Cromafines/fisiología , Vesículas Secretoras/fisiología , Aminas/metabolismo , Análisis de Varianza , Animales , Bovinos , Células Cromafines/ultraestructura , Gránulos Cromafines/ultraestructura , Simulación por Computador , Microscopía Confocal/métodos , Microscopía Electrónica de Rastreo/métodos , Modelos Biológicos , Vesículas Secretoras/ultraestructura , Factores de TiempoRESUMEN
Amperometry is a simple and powerful technique to study exocytosis at the single cell level. By positioning and polarizing (at an appropriate potential at which the molecules released by the cell can be oxidized) a carbon fiber microelectrode at the top of the cell, each exocytotic event is detected as an amperometric spike. More particularly, a portion of these spikes has previously been shown to present a foot, i.e. a small pedestal of current that precedes the spike itself. Among the important number of works dealing with the monitoring of exocytosis by amperometry under different conditions, only a few studies focus on amperometric spikes with a foot. In this work, by coupling our previous and recent experiments on chromaffin cells (that release catecholamines after stimulation) with literature data, we bring more light on what an amperometric foot and particularly its features, represents.
Asunto(s)
Células Cromafines/fisiología , Gránulos Cromafines/fisiología , Conductometría/métodos , Exocitosis , Vesículas Secretoras/fisiología , Animales , Bovinos , Células Cultivadas , Células Cromafines/química , Células Cromafines/ultraestructura , Gránulos Cromafines/química , Vesículas Secretoras/químicaRESUMEN
Bovine chromaffin granules were exposed to different isotonic non-ionic and ionic solutions (sucrose; Ca2+- and Mg2+-free phosphate-buffered saline; Tris-HCl + NaCl; Ca2+- and Mg2+-free phosphate-buffered saline + sucrose; Tris-HCl + sucrose) at pH 7 and then frozen either in suspension or as firm pellets. Freezing was performed without prefixation or antifreeze treatments either by 'standard' techniques (approx. 1 mm3 suspended or pelleted material on gold specimen supports dipped into liquid Freon) or with increased cooling rates by spraying suspensions into liquid propane ('spray-freezing'). Regardless of the freezing method, membrane-intercalated particles were always randomly distributed when chromaffin granules were frozen in suspension. In contrast, forced physical contact between granules produced by centrifugation (12000 X g, 25 min) provoked dispersal of membrane-intercalated particles, but only in the presence of ions. Sucrose or EDTA in an ionic environment had no inhibitory effect. The following conclusions are derived: (1) Even below the reported phase transition region particle clustering is possible. (2) Chromaffin granule membranes are not liable to thermotropic segregation of membrane-intercalated particles. (3) Although the low freezing rates of 'standard' freezing techniques produce large-scale segregation artefacts (by which suspended chromaffin granules are pushed together within the segregated solute) this does not result in intramembraneous particle segregation. (4) Forced physical contact produces a Ca2+-independent particle segregation, but only when repulsive electrostatic forces of membrane components are partially screened in an ionic environment. (5) This does not invalidate results obtained by others, showing Ca2+-mediated chromaffin granules agglomeration and segregation of membrane-intercalated particles, but it might indicate the occurrence of another, not directly Ca2+-dependent particle segregation mechanism in a prefusional stage of close membrane-to-membrane contact during exocytosis.
Asunto(s)
Gránulos Cromafines/fisiología , Sistema Cromafín/fisiología , Animales , Calcio , Bovinos , Centrifugación , Fenómenos Químicos , Química Física , Gránulos Cromafines/ultraestructura , Exocitosis , Técnica de Fractura por Congelación , Fluidez de la Membrana , MétodosRESUMEN
In chromaffin cells of the adrenal medulla, catecholamines are stored in secretory granules. Different methods have been described to purify chromaffin granules. In the present study, storage granules were prepared using isoosmotic self-generating Percoll gradients or hyperosmotic sucrose gradients, and a comparison of their physical properties in response to osmotic changes was made. Catecholamines, dopamine beta-hydroxylase activity and protein were detected both in the external medium and in the granule fraction according to the medium osmolality. Suspension turbidity was used as a measure of organelle integrity. Acetylcholinesterase activity was found to be associated with both isoosmotically and hyperosomotically prepared granules. The total acetylcholinesterase activity was determined after adding Triton X-100 to the assay medium. When adrenal medullary tissue was homogenized in buffers containing echothiopate, an inhibitor of acetylcholinesterase, only 15-20% of enzyme activity was inhibited, excluding the possibility that main granule acetylcholinesterase could be due to contamination by plasma membrane fragments, endoplasmic reticulum and Golgi membranes. When granules were suspended in hypoosmotic buffers, a soluble acetylcholinesterase form was released into the external medium, while an insoluble acetylcholinesterase form was still found associated with the membrane fraction. Soluble acetylcholinesterase was found to be released differently than soluble dopamine beta-hydroxylase, indicating that acetylcholinesterase may be associated with a more osmotically resistant granule population.
Asunto(s)
Acetilcolinesterasa/análisis , Gránulos Cromafines/fisiología , Sistema Cromafín/fisiología , Animales , Catecolaminas/metabolismo , Bovinos , Fraccionamiento Celular , Inhibidores de la Colinesterasa/farmacología , Gránulos Cromafines/enzimología , Dopamina beta-Hidroxilasa/metabolismo , Concentración OsmolarRESUMEN
Carbon-13 spin-lattice relaxation times, T1, have been measured in whole adrenal medullary tissue slices, in suspensions of isolated chromaffin granules, in the reconcentrated chromaffin granule lysate, and in various model solutions containing catecholamines. ATP, chromogranins and Ca2+. Reorientational correlation times have been calculated at 10 degrees C using T1 data and nuclear Overhauser enhancements for protonated carbons on both catecholamines and nucleotides. Correlation times in all media are relatively short and characteristic of highly fluid aqueous phases. Adrenalin and ATP exhibit substantial differences in correlation times in all media, however, the ratio tau R (ATP): tau R(catecholamine) ranging from 2.4 in simple 3:1 adrenalin-ATP solutions to 4 in intact chromaffin granules. This difference, as well as the relatively high absolute reorientational mobilities of both components, confirms the importance of labile ionic interactions between ATP and catecholamines, but rules out the presence of high concentrations of base-stacked structures. Participation of the chromogranins in ternary complexes with catecholamines and ATP appears to be of minor importance. Ionic interactions to the protein are not reflected in either 13C T1 values or chemical shifts of arginine or glutamate sidechain resonances, or in the 13C chemical shifts of ATP or catecholamines. Very labile protein-ATP binding appears to be reflected in the correlation time measurements, however, which show selective immobilization of ATP relative to catecholamine in the presence of soluble protein. Osmotic measurements indicate that solutions containing adrenaline, ATP and Ca2+ are highly nonideal, but probably not sufficiently so to account fully for the osmotic stabilization of the chromaffin through their polyelectrolyte properties, exert a significant influence on the intragranular osmolality. The osmotic lowering due to polyion-counterion interactions has been estimated semiquantitatively using a theory developed by Oosawa.
Asunto(s)
Médula Suprarrenal/fisiología , Gránulos Cromafines/fisiología , Sistema Cromafín/fisiología , Adenosina Trifosfato/análisis , Animales , Calcio , Catecolaminas/análisis , Bovinos , Epinefrina/análisis , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Concentración OsmolarRESUMEN
The dye, oxonol-V (bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol), can be used to estimate the transmembrane potential of chromaffin granules. The potentials result either from a resting-state Donnan equilibrium (inside negative at pH 6.6) or from an ATP-driven proton pump. The fluorescence and absorption changes generated by ATP addition depended on the pH of the medium and the dye-to-vesicle ratio. Energization resulted in an increase in the number of oxonol-V binding sites, the new binding sites having the same dissociation constant. The rate of dye association was higher with resting than with energized chromaffin granules. The absorption change was associated with a red shift whereas the fluorescence change involved a quenching due to the increase in dye concentration on the membrane. The absorption and fluorescence changes varied linearly with the transmembrane potential difference when the interior potential was positive relative to the medium.
Asunto(s)
Alquenos , Gránulos Cromafines/fisiología , Sistema Cromafín/fisiología , Colorantes Fluorescentes , Isoxazoles , Potenciales de la Membrana , Oxazoles , Absorción , Animales , Transporte Biológico Activo , Bovinos , Gránulos Cromafines/metabolismo , Técnicas In Vitro , Norepinefrina/metabolismoRESUMEN
The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K+. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10 microM forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+]i), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+]i. This additional pathway may be the target for cAMP action.
Asunto(s)
Médula Suprarrenal/metabolismo , Carbacol/farmacología , Catecolaminas/metabolismo , Gránulos Cromafines/fisiología , Sistema Cromafín/fisiología , AMP Cíclico/fisiología , Potasio/farmacología , Médula Suprarrenal/efectos de los fármacos , Animales , Bovinos , Cinética , Nicotina/farmacologíaRESUMEN
The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane-permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3-(5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.