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1.
Sci Rep ; 10(1): 7540, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371955

RESUMO

Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.


Assuntos
Medula Suprarrenal/fisiologia , Técnicas Citológicas/métodos , Animais , Bovinos , Fracionamento Celular , Grânulos Cromafim/metabolismo , Fusão de Membrana , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo
2.
J Am Chem Soc ; 142(9): 4093-4097, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32069039

RESUMO

We have developed the means to simultaneously measure the physical size and count catecholamine molecules in individual nanometer transmitter vesicles. This is done by combining resistive pulse (RP) measurements in a nanopore pipet and vesicle impact electrochemical cytometry (VIEC) at an electrode as the vesicle exits the nanopore. Analysis of freshly isolated bovine adrenal vesicles shows that the size and internal catecholamine concentration of vesicles varies with the occurrence of a dense core inside the vesicles. These results might benefit the understanding about the vesicles maturation, especially involving the "sorting by retention" process and concentration increase of intravesicular catecholamine. The methodology is applicable to understanding soft nanoparticle collisions on electrodes, vesicles in exocytosis and phagocytosis, intracellular vesicle transport, and analysis of electroactive drugs in exosomes.


Assuntos
Catecolaminas/análise , Grânulos Cromafim/química , Técnicas Eletroquímicas/métodos , Nanoporos , Animais , Bovinos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Tamanho da Partícula
3.
J Neurochem ; 154(6): 598-617, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32058590

RESUMO

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.


Assuntos
Grânulos Cromafim/fisiologia , Receptores de Detecção de Cálcio/fisiologia , Sinaptotagminas/genética , Sinaptotagminas/fisiologia , Acetilcolina/farmacologia , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Fenômenos Eletrofisiológicos , Exocitose , Feminino , Cinética , Masculino , Fusão de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células PC12 , Ratos , Proteínas SNARE/metabolismo , Frações Subcelulares/metabolismo , Sinaptotagmina I/fisiologia
4.
J Neurochem ; 152(3): 299-314, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31677273

RESUMO

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild-type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt-KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (-30%) and Imax (-21%) of granules in KO cells relative to the wild-type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.


Assuntos
Células Cromafins/metabolismo , Grânulos Cromafim/metabolismo , Epinefrina/metabolismo , Norepinefrina/metabolismo , Animais , Exocitose/fisiologia , Masculino , Camundongos , Camundongos Knockout , Feniletanolamina N-Metiltransferase/deficiência , Feniletanolamina N-Metiltransferase/genética
5.
Sci Rep ; 9(1): 18471, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804600

RESUMO

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells.


Assuntos
Grânulos Cromafim/efeitos dos fármacos , Cloridrato de Fingolimode/toxicidade , Esclerose Múltipla/tratamento farmacológico , Células Neuroendócrinas/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Grânulos Cromafim/metabolismo , Grânulos Cromafim/patologia , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Células Neuroendócrinas/citologia , Células Neuroendócrinas/metabolismo , Cultura Primária de Células , Proteína 25 Associada a Sinaptossoma/metabolismo , Testes de Toxicidade
6.
Pflugers Arch ; 470(1): 125-134, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28951968

RESUMO

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.


Assuntos
Grânulos Cromafim/metabolismo , Técnicas Eletroquímicas/métodos , Exocitose , Potenciais de Ação , Animais , Grânulos Cromafim/fisiologia , Citofotometria/instrumentação , Citofotometria/métodos , Técnicas Eletroquímicas/instrumentação , Humanos
7.
Pflugers Arch ; 470(1): 181-186, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28730385

RESUMO

Actin is one of the most ubiquitous protein playing fundamental roles in a variety of cellular processes. Since early in the 1980s, it was evident that filamentous actin (F-actin) formed a peripheral cortical barrier that prevented vesicles to access secretory sites in chromaffin cells in culture. Later, around 2000, it was described that the F-actin structure accomplishes a dual role serving both vesicle transport and retentive purposes and undergoing dynamic transient changes during cell stimulation. The complex role of the F-actin cytoskeleton in neuroendocrine secretion was further evidenced when it has been proved to participate in the scaffold structure holding together the secretory machinery at active sites and participate in the generation of mechanical forces that drive the opening of the fusion pore, during the first decade of the present century. The complex vision of the multiple roles of F-actin in secretion we have acquired to date comes largely from studies performed on traditional 2D cultures of primary cells; however, recent evidences suggest that these may not accurately mimic the 3D in vivo environment, and thus, more work is now needed on adrenomedullary cells kept in a more "native" configuration to fully understand the role of F-actin in regulating chromaffin granule transport and secretion under physiological conditions.


Assuntos
Actinas/metabolismo , Grânulos Cromafim/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Exocitose , Humanos , Via Secretória
8.
Pflugers Arch ; 470(1): 135-141, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28779472

RESUMO

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.


Assuntos
Catecolaminas/metabolismo , Grânulos Cromafim/metabolismo , Exocitose , Animais , Grânulos Cromafim/fisiologia , Cromograninas/metabolismo , Técnicas Eletroquímicas/métodos , Humanos , Transmissão Sináptica
9.
Pflugers Arch ; 470(1): 7-11, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28801866

RESUMO

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.


Assuntos
Grânulos Cromafim/fisiologia , Neurossecreção , Animais , Grânulos Cromafim/metabolismo , Humanos , Transmissão Sináptica
10.
Pflugers Arch ; 470(1): 1-6, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29110079

RESUMO

The chromaffin cells (CCs) of the adrenal medulla play a key role in the control of circulating catecholamines to adapt our body function to stressful conditions. A huge research effort over the last 35 years has converted these cells into the Escherichia coli of neurobiology. CCs have been the testing bench for the development of patch-clamp and amperometric recording techniques and helped clarify most of the known molecular mechanisms that regulate cell excitability, Ca2+ signals associated with secretion, and the molecular apparatus that regulates vesicle fusion. This special issue provides a state-of-the-art on the many well-known and unsolved questions related to the molecular processes at the basis of CC function. The issue is also the occasion to highlight the seminal work of Antonio G. García (Emeritus Professor at UAM, Madrid) who greatly contributed to the advancement of our present knowledge on CC physiology and pharmacology. All the contributors of the present issue are distinguished scientists who are either staff members, external collaborators, or friends of Prof. García.


Assuntos
Medula Suprarrenal/metabolismo , Grânulos Cromafim/metabolismo , Transdução de Sinais , Medula Suprarrenal/citologia , Animais , Humanos
11.
Pflugers Arch ; 470(1): 155-167, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28852855

RESUMO

The extent and type of hormones and active peptides secreted by the chromaffin cells of the adrenal medulla have to be adjusted to physiological requirements. The chromaffin cell secretory activity is controlled by the splanchnic nerve firing frequency, which goes from approximately 0.5 Hz in basal conditions to more than 15 Hz in stress. Thus, these neuroendocrine cells maintain a tonic release of catecholamines under resting conditions, massively discharge intravesicular transmitters in response to stress, or adequately respond to moderate stimuli. In order to adjust the secretory response to the stimulus, the adrenal chromaffin cells have an appropriate organization of Ca2+ channels, secretory granules pools, and sets of proteins dedicated to selectively control different steps of the secretion process, such as the traffic, docking, priming and fusion of the chromaffin granules. Among the molecules implicated in such events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Ca2+ sensors like Munc13 and synaptotagmin-1, chaperon proteins such as Munc18, and the actomyosin complex. In the present review, we discuss how these different actors contribute to the extent and maintenance of the stimulus-dependent exocytosis in the adrenal chromaffin cells.


Assuntos
Medula Suprarrenal/metabolismo , Grânulos Cromafim/metabolismo , Exocitose , Animais , Canais de Cálcio/metabolismo , Humanos , Proteínas de Transporte Vesicular/metabolismo
12.
Anal Biochem ; 536: 1-7, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28760673

RESUMO

The study of chromaffin secretory vesicles (SVs) has contributed immensely to our understanding of exocytosis. These organelles, also called chromaffin granules, are a specific type of large dense secretory vesicle found in many endocrine cells and neurons. Traditionally, they have been isolated from bovine adrenal glands due to the large number of SVs that can be obtained from this tissue. However, technical advances now make it possible to obtain very pure preparations of SVs from mice, which is particular interesting for functional studies given the availability of different genetically modified strains of mice. Despite the small size of the mouse adrenal medulla (400-500 µm and less than 2 mg in weight), we have successfully carried out functional studies on SVs isolated from WT and knockout mice. As such, we present here our method to purify crude vesicles and to fractionate mouse chromaffin SVs, along with examples of their functional characterization.


Assuntos
Grânulos Cromafim/metabolismo , Vesículas Secretórias/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vesículas Secretórias/química
13.
J Gen Physiol ; 149(8): 755, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28716806

RESUMO

JGP study shows how synaptotagmins 1 and 7 affect chromaffin cell granule fusion.


Assuntos
Células Cromafins , Acidente Vascular Cerebral , Grânulos Cromafim , Humanos , Cinética , Isoformas de Proteínas
14.
Biophys J ; 113(6): 1251-1259, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28400045

RESUMO

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.


Assuntos
Grânulos Cromafim/metabolismo , Lipossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Animais , Difusão , Escherichia coli , Recuperação de Fluorescência Após Fotodegradação , Bicamadas Lipídicas/metabolismo , Microscopia , Modelos Biológicos , Ratos , Fatores de Tempo
15.
J Neurochem ; 137(6): 897-903, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26990968

RESUMO

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).


Assuntos
Exocitose/fisiologia , Neurotransmissores/metabolismo , Vesículas Secretórias/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Grânulos Cromafim/fisiologia , Cromograninas/metabolismo , Humanos , Modelos Biológicos
16.
J Neurochem ; 137(6): 860-6, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26843469

RESUMO

In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss the role of transport systems and the existence of an F-actin cortical structure as the main factors behind the formation of organelle subpopulations in this neuroendocrine cell model. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015). Cover image for this issue: doi: 10.1111/jnc.13322.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/fisiologia , Células Cromafins/ultraestrutura , Organelas/fisiologia , Animais , Grânulos Cromafim , Humanos
17.
Cell Tissue Res ; 363(3): 693-712, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26572539

RESUMO

Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell.


Assuntos
Catecolaminas/metabolismo , Grânulos Cromafim/metabolismo , Cromogranina A/deficiência , Metabolismo Energético , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Animais , Western Blotting , Grânulos Cromafim/efeitos dos fármacos , Grânulos Cromafim/ultraestrutura , Cromogranina A/metabolismo , Dopamina/metabolismo , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Epinefrina/metabolismo , Exocitose/efeitos dos fármacos , Glucose/metabolismo , Glicogênio/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Insulina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Norepinefrina/metabolismo , Nervos Esplâncnicos/efeitos dos fármacos , Nervos Esplâncnicos/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo
18.
Nat Struct Mol Biol ; 22(10): 815-23, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26389740

RESUMO

The Ca(2+) sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg(2+) completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca(2+). Altogether, our data indicate that at physiological ion concentrations Ca(2+)-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.


Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinaptotagmina I/química , Sinaptotagmina I/metabolismo , Animais , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Grânulos Cromafim/metabolismo , Cromatografia por Troca Iônica , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Modelos Teóricos , Técnicas de Patch-Clamp , Conformação Proteica , Ratos , Proteínas SNARE/metabolismo , Análise Espectral
19.
Eur Neuropsychopharmacol ; 25(10): 1753-66, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26187454

RESUMO

Stress has been considered determinant in the etiology of depression. The adrenal medulla plays a key role in response to stress by releasing catecholamines, which are important to maintain homeostasis. We aimed to study the adrenal medulla in a mouse model of depression induced by 21 days of unpredictable chronic stress (UCS). We observed that UCS induced a differential and time-dependent change in adrenal medulla. After 7 days of UCS, mice did not show depressive-like behavior, but the adrenal medullae show increased protein and/or mRNA levels of catecholamine biosynthetic enzymes (TH, DßH and PNMT), Neuropeptide Y, the SNARE protein SNAP-25, the catecholamine transporter VMAT2 and the chromaffin progenitor cell markers, Mash1 and Phox2b. Moreover, 7 days of UCS induced a decrease in the chromaffin progenitor cell markers, Sox9 and Notch1. This suggests an increased capacity of chromaffin cells to synthesize, store and release catecholamines. In agreement, after 7 days, UCS mice had higher NE and EP levels in adrenal medulla. Opposite, when mice were submitted to 21 days of UCS, and showed a depressive like behavior, adrenal medullae had lower protein and/or mRNA levels of catecholamine biosynthetic enzymes (TH, DßH, PNMT), catecholamine transporters (NET, VMAT1), SNARE proteins (synthaxin1A, SNAP25, VAMP2), catecholamine content (EP, NE), and lower EP serum levels, indicating a reduction in catecholamine synthesis, re-uptake, storage and release. In conclusion, this study suggests that mice exposed to UCS for a period of 21 days develop a depressive-like behavior accompanied by an impairment of adrenal medullary function.


Assuntos
Medula Suprarrenal/fisiopatologia , Transtorno Depressivo/fisiopatologia , Estresse Psicológico/fisiopatologia , Medula Suprarrenal/patologia , Animais , Peso Corporal , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Catecolaminas/metabolismo , Grânulos Cromafim/fisiologia , Doença Crônica , Corticosterona/sangue , Transtorno Depressivo/patologia , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Tamanho do Órgão , RNA Mensageiro/metabolismo , Células-Tronco/fisiologia , Estresse Psicológico/patologia , Incerteza
20.
Neuroscience ; 295: 117-25, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25813703

RESUMO

Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles.


Assuntos
Células Cromafins/citologia , Grânulos Cromafim/fisiologia , Exocitose/fisiologia , Esfingomielinas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/fisiologia , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Grânulos Cromafim/efeitos dos fármacos , Grânulos Cromafim/ultraestrutura , Capacitância Elétrica , Exocitose/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia Eletrônica , Técnicas de Patch-Clamp , Proteínas SNARE/metabolismo , Esfingomielina Fosfodiesterase/farmacologia , Estatísticas não Paramétricas , Proteína 25 Associada a Sinaptossoma/genética , Transfecção
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