RESUMO
AIMS: Microglia survey the brain environment by sensing alarm signals to provide the first line of defense against injury or infection after which they acquire an activated phenotype, but they also respond to chemical signals sent from brain mast cells, sentinels of the immune system, when these are degranulated in response to noxious agents. Nevertheless, excessive microglia activation damages the surrounding healthy neural tissue causing progressive loss of neurons and inducing chronic inflammation. Thus, it would be of intense interest the development and application of agents which prevent mast cell mediator release and inhibit the actions of such mediators once released on microglia. MAIN METHODS: Fluorescence measurements of fura-2 and quinacrine were used to measure intracellular Ca2+ signaling and exocytotic vesicle fusion in resting and activated microglia. KEY FINDINGS: We show that treatment of microglia with a cocktail of mast cell mediators induces microglia activation, phagocytosis, and exocytosis, and reveal by the first-time microglia undergo a phase of vesicular acidification just before the exocytotic fusion occurs. This acidification is an important process for vesicular maturation and contributes with â¼25 % to the content that the vesicle can store and later release by exocytosis. Pre-incubation with ketotifen, a mast cell stabilizer and H1R antagonist completely abolished histamine-mediated calcium signaling and acidification of microglial organelles, and concomitantly reduced the discharge of vesicle contents. SIGNIFICANCE: These results highlight a key role for vesicle acidification in microglial physiology and provide a potential therapeutic target for diseases related to mast cell and microglia-mediated neuroinflammation.
Assuntos
Cetotifeno , Microglia , Encéfalo , Vesículas Secretórias , Concentração de Íons de HidrogênioRESUMO
Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.
Assuntos
Neurônios/metabolismo , Neurotransmissores/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Exocitose , Humanos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Regulação para Cima , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
BACKGROUND/AIMS: Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored. METHODS: Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca2+]i and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques. RESULTS: Local application onto microglia of the MC mediator cocktail elicited Ca2+ transients and exocytotic release associated with quinacrine dye de-staining. Ca2+ signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca2+ transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression. CONCLUSION: Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Comunicação Celular , Histamina/metabolismo , Mastócitos/metabolismo , Microglia/metabolismo , Animais , Ratos , Ratos WistarRESUMO
Hepatocellular carcinoma (HCC) is the sixth most common neoplasia and the fourth most common cause of cancer-related mortality worldwide. Sorafenib is the first-line molecular therapy for patients in an advanced stage of HCC. However, the recommended clinical dose of Sorafenib is associated with several complications, which derive from its lack of cell specificity and its very low water solubility. To circumvent these drawbacks, in the present study we developed two sugar-coated polydiacetylene-based nanomicelles-Sorafenib carriers targeting mannose and asialoglycoprotein receptors (MR and ASGPR, respectively). The strategies allowed the inducement of apoptosis and reduction of cell proliferation at a nanomolar, instead of micromolar, range in liver cancer cells. The study showed that, contrary to literature data, Sorafenib included into the pMicMan (Man = mannose) vector (targeting MR) is more efficient than pMicGal (Gal = galactose) (targeting ASGPR). Indeed, pMicMan increased the endosomal incorporation with an increased intracellular Sorafenib concentration that induced apoptosis and reduced cell proliferation at a low concentration range (10-20 nM).
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Galactose/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Manose/administração & dosagem , Nanopartículas/administração & dosagem , Polímero Poliacetilênico/administração & dosagem , Sorafenibe/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Endossomos/metabolismo , Galactose/química , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Manose/química , Receptor de Manose/metabolismo , Micelas , Nanopartículas/química , Polímero Poliacetilênico/química , Sorafenibe/químicaRESUMO
BACKGROUND: Mast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions. METHODS: Calcium imaging was used to simultaneously record changes in intracellular calcium [Ca2+]i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat. RESULTS: Bidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca2+]i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca2+]i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS). CONCLUSIONS: MC-hippocampal neuron interaction affects neuronal [Ca2+]i and exocytosis signaling through a NMDAR-dependent mechanism.
Assuntos
Comunicação Celular/fisiologia , Hipocampo/metabolismo , Mastócitos/metabolismo , Neurônios/metabolismo , Proteoglicanas/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Técnicas de Cocultura , Hipocampo/química , Hipocampo/citologia , Mastócitos/química , Neurônios/química , Proteoglicanas/análise , RatosRESUMO
To ensure normal immune function, mast cells employ different pathways to release mediators. Here, we report a thus far unknown capacity of mast cells to recycle and reuse secretory granules after an antigen-evoked degranulation process under physiological conditions; this phenomenon involves the existence of a recycling secretory granule pool that is available for release in a short time scale. Rapid endocytic modes contributed to the recycling of â¼60% of the total secretory granule population, which involved kiss-and-run and cavicapture mechanisms, causing retention of the intragranular matrix. We found the presence of normal-size granules and giant actomyosin- and dynamin-dependent granules, which were characterized by large quantal content. These large structures allowed the recovered mast cells to release a large amount of 5-HT, compensating for the decrease in the number of exocytosed secretory granules. This work uncovers a new physiological role of the exo-endocytosis cycle in the immunological plasticity of mast cells and reveals a new property of their biological secretion.
Assuntos
Degranulação Celular , Imunoglobulina E/metabolismo , Mastócitos/fisiologia , Fusão de Membrana , Vesículas Secretórias/metabolismo , Actinas/metabolismo , Animais , Antígenos/metabolismo , Calcimicina/farmacologia , Degranulação Celular/efeitos dos fármacos , Dinaminas/metabolismo , Técnicas Eletroquímicas , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Miosina Tipo II/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Serotonina/metabolismoRESUMO
5-hydroxytriptamine (5-HT, serotonin) storage and release in mast cell (MC) secretory granules (SG) are dependent on serglycin proteoglycans (PG). This notion is based on the studies of MC of the connective tissue subtype that predominantly contain PG of the heparin type, whereas intestinal mucosal MC, which contain predominantly chondroitin sulfate, have been poorly explored. In the present study, we addressed the possibility that PG contents may differently affect the storage and release of preformed mediators in these two MC subclasses and explain in part their different functional properties. Rat peritoneal (PMC) and intestinal mast cells (IMC) were isolated and purified using a percoll gradient, and the efflux of 5-HT from each SG was measured by amperometric detection. IMC exhibited a â¼34% reduction in the release of 5-HT compared with PMC because of a lower number of exocytotic events, rather than a lower secretion per single exocytotic event. Amperometric spikes from IMC exhibited a slower decay phase and increased half-width but a similar ascending phase and foot parameters, indicating that the fusion pore kinetics are comparable in both MC subclasses. We conclude that both PG subtypes are equally efficient systems, directly involved in serotonin accumulation, and play a crucial role in regulating the kinetics of exocytosis from SG, providing specific secretory properties for the two cellular subtypes.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Mastócitos/metabolismo , Peritônio/citologia , Peritônio/metabolismo , Serotonina/metabolismo , Animais , Células Cultivadas , Exocitose/fisiologia , Cinética , Mastócitos/citologia , Taxa de Depuração Metabólica , Especificidade de Órgãos/fisiologia , Ratos , Ratos WistarRESUMO
Secretory granules (SGs) of mast cells (MCs) release their contents to mediate many biological events and a variety of inflammatory diseases and have important protective roles in innate host defense and pathological functions in allergic reactions and anaphylaxis. There are two modes of MC degranulation during the release of granule contents to the extracellular environment. Anaphylactic degranulation (AND) after IgE-mediated activation is characterized by a rapid swelling and fusion of MC granules as well as abrupt mediators release. Piecemeal degranulation (PMD) is a slow and selective secretion of distinct granule mediators by vesicles shuttling from the granule compartment to the plasma membrane, and it is associated with several chronic diseases. Following degranulation, endocytosis is a fundamental biological event that is necessary to recycle granules and maintain the secretory response during repetitive stimulation. Rapid endocytosis through transient fusion (kiss-and-run, cavicapture and compound exo-endocytosis) has been described in MCs and can also result in the selective release of granule contents. In summary, several possible exo-endocytic mechanisms control the kinetics and magnitude of transmitter release, and each mechanism is associated with a different impact on granule replenishment, cell recovery, and consequently MC function under both normal and pathological conditions.
RESUMO
The key role of mast cells (MC), either in development of inflammatory pathologies or in response to environmental stress, has been widely reported in recent years. Previous studies have described the effects of corticotropin-releasing hormone (CRH), which is released from inflamed tissues by cellular stress signals, on MC degranulation, a process possibly driven by selective secretion of mediators (piecemeal degranulation). In this study, we introduce a novel granular exo-endocytic pathway induced by CRH on peritoneal MC. We found that CRH triggers substantial exocytosis, which is even stronger than that induced by Ag stimulation and is characterized by large quantal size release events. Membrane fluorescence increases during stimulation in the presence of FM1-43 dye, corroborating the strength of this exocytosis, given that discrete upward fluorescence steps are often observed and suggesting that secretory granules are preferentially released by compound exocytosis. Additionally, the presence of a depot of large tubular organelles in the cytoplasm suggests that the exocytotic process is tightly coupled to a fast compound endocytosis. This CRH-stimulated mechanism is mediated through activation of adenylate cyclase and an increase of cAMP and intracellular Ca(2+), as evidenced by the fact that the effect of CRH is mimicked by forskolin and 8-bromo-cAMP. Thus, these outcomes constitute new evidence for the critical role of MC in pathophysiological conditions within a cellular stress environment and an alternative membrane trafficking route mediated by CRH.
Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Mastócitos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Mastócitos/fisiologia , Camundongos Endogâmicos C57BL , Microscopia Confocal , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Ratos Wistar , Vesículas Secretórias/metabolismoRESUMO
An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of â¼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.
Assuntos
Miosina Tipo II/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Miosina Tipo II/antagonistas & inibidores , Ligação Proteica , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Vesículas Transportadoras/efeitos dos fármacosRESUMO
In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified "large-capacitance flickers" that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent "compound cavicapture," most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.
Assuntos
Membrana Celular/metabolismo , Endocitose , Exocitose , Mastócitos/fisiologia , Vesículas Secretórias/metabolismo , Animais , Células Cultivadas , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Fusão de Membrana , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Compostos de Piridínio/química , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Imagem com Lapso de Tempo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
In chromaffin cells, Ca(2+) binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca(2+) and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca(2+) binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca(2+) binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca(2+) binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.
Assuntos
Cálcio/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Camundongos Knockout/metabolismo , Sinaptotagminas/metabolismo , Animais , Células Cromafins/citologia , Feminino , Masculino , Camundongos , Camundongos Knockout/genética , Estrutura Terciária de Proteína , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sinaptotagminas/genéticaRESUMO
Although endocytosis involves the fission pore, a transient structure that produces the scission between vesicle and plasma membranes, the dimensions and dynamics of fission pores remain unclear. Here we report that the pore resistance changes proceed in three distinct phases: an initial phase where the resistance increases at 31.7 ± 2.9 GΩ/second, a slower linear phase with an overall slope of 11.7 ± 1.9 GΩ/second and a final increase in resistance more steeply (1189 ± 136 GΩ/second). The kinetics of these changes was calcium dependent. These sequential stages of the fission pore may be interpreted in terms of pore geometry as changes, first in pore diameter and then in pore length, according to which, before fission, the pore diameter consistently decreased to a value near 4 nm, whereas the pore length ranged between 20 and 300 nm. Dynamin, a mechanochemical GTPase, plays an important role in accelerating the fission event, preferentially in endocytotic vesicles of regular size, by increasing the rates of pore closure during the first and second phases of the fission pore, but hardly affected larger and longer-lived endocytotic events. These results suggest that fission pores are dynamic structures that form thin and long membrane necks regulated by intracellular calcium. Between calcium mediators, dynamin functions as a catalyst to increase the speed of single vesicle endocytosis.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Endocitose , Animais , Impedância Elétrica , Mastócitos/metabolismo , Camundongos , PorosidadeRESUMO
During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites.
Assuntos
Exocitose , Miosina Tipo II/fisiologia , Animais , Transporte Biológico , Catecolaminas/metabolismo , Permeabilidade da Membrana Celular , Células Cromafins/química , Células Cromafins/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Fusão de Membrana , Modelos Biológicos , Método de Monte Carlo , Miosina Tipo II/química , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/químicaRESUMO
Galantamine is a drug in clinical use for the treatment of Alzheimer's disease, but its mechanism(s) of action remains controversial. Here we addressed the question whether galantamine could potentiate neurotransmitter release by inhibiting small conductance Ca2+ -activated K+ channels (KCa2). Galantamine potentiated catecholamine secretory responses induced by 10 s pulses of acetylcholine and high [K+]o applied to fast-superfused bovine adrenal chromaffin cell populations. Catecholamine release was significantly enhanced by galantamine although we did not find concentration dependence in the range 0.1-1 microM. The KCa2 channel blocker apamin (0.3 microM) occluded the potentiating effects of galantamine on acetylcholine-evoked secretion. Like apamin, galantamine also modified the firing of action potentials, but to a lesser extent. In addition, 1 microM galantamine reduced by 41% the KCa2 current without modifying the voltage-dependent Ca2+ currents. These results constitute the first direct evidence that galantamine can potentiate neurotransmitter release by blocking KCa2 channels, in addition to its already demonstrated capacity to mildly block acetylcholinesterase or potentiate allosterically nicotinic receptors.
Assuntos
Catecolaminas/metabolismo , Células Cromafins/efeitos dos fármacos , Galantamina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Medula Suprarrenal/citologia , Animais , Apamina/farmacologia , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Células Cromafins/fisiologia , Masculino , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologiaRESUMO
We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.
Assuntos
Astrócitos/fisiologia , Células Cromafins/fisiologia , Exocitose/fisiologia , Neurônios/fisiologia , Animais , Astrócitos/citologia , Astrócitos/ultraestrutura , Sinalização do Cálcio , Bovinos , Células Cultivadas , Córtex Cerebral/fisiologia , Células Cromafins/citologia , Meios de Cultivo Condicionados , Eletrofisiologia , Neurônios/citologia , RatosRESUMO
In bovine chromaffin cells fast-superfused with Krebs-HEPES solution containing 1-2 mM Ca2+, 5 s pulses of choline (1-10 mM), elicited catecholamine secretory responses that were only approximately 10% of those evoked by ACh (0.01-0.1 mM). However, in high-Ca2+ solutions (10-20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine alpha7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing alpha3beta4, however, the inward current elicited by choline amounted to only 4% of the size of alpha7 current. Our results suggest that choline activates the entry of Ca2+ through alpha7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+-dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high-Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Catecolaminas/metabolismo , Colina/farmacologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Atropina/farmacologia , Cálcio/farmacologia , Bovinos , Condutividade Elétrica , Mecamilamina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Potássio/farmacologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Xenopus laevisRESUMO
Galantamine is currently used to treat Alzheimer's disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Apoptose/efeitos dos fármacos , Fura-2/análogos & derivados , Galantamina/farmacologia , Fragmentos de Peptídeos/toxicidade , Receptores Nicotínicos/metabolismo , Tapsigargina/toxicidade , Análise de Variância , Animais , Western Blotting/métodos , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Bovinos , Linhagem Celular Tumoral , Inibidores da Colinesterase/farmacologia , Células Cromafins/efeitos dos fármacos , Interações Medicamentosas , Inibidores Enzimáticos/toxicidade , Citometria de Fluxo/métodos , Fura-2/metabolismo , Humanos , Imuno-Histoquímica/métodos , Neuroblastoma , Nicotina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RatosRESUMO
The ether-a-go-go-related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike-frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single-cell level by means of perforated patch-clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N-methyltransferase and ERG channels suggested expression of these channels in epinephrine- but not in norepinephrine-containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long-QT running heart.