RESUMEN
Cationic heterocycles, an important class of organic compounds soluble in polar solvents, have been gaining attention in the construction of fluorescent probes. This paper reports the quick synthesis of novel pyrido[1',2';2,3]imidazo[5,1-a]isoquinoliniums starting from 2-(2-ethynylphenyl)imidazo[1,2-a]pyridines at room temperature via intramolecular cyclization by employing a catalytic amount of silver trifluoromethanesulfonate in addition to lithium trifluoromethanesulfonate and silica gel as the counter anion source and additive, respectively. The designed pyridoimidazoisoquinoliniums consisted of an imidazo[1,2-a]pyridine fused isoquinolinium. The X-ray diffraction results revealed that pyrido[1',2';2,3]imidazo[5,1-a]isoquinolinium trifluoromethanesulfonate contained considerable planar parent skeletons and interacted by π-π stacking with neighbouring molecules. Furthermore, in a methanol solution the designed 6-phenyl derivative exhibited strong fluorescence in the 420-450 nm region in addition to strong mitochondrial specificity in a cell staining assay.
RESUMEN
Aggregation of IgE bound to the high-affinity IgE receptor (FcεRI) by a multivalent antigen induces mast cell activation, while disaggregation of aggregated FcεRI by monomer hapten immediately terminates degranulation mediated by dephosphorylation of Syk and mediates a decrease in intracellular Ca2+ concentration ([Ca2+]i). The actin polymerization state is intimately involved in mast cell activation mediated by FcεRI aggregation. However, the relation between aggregation-disaggregation of FcεRI and actin rearrangement in mast cells is not well understood. The addition of a multivalent antigen rapidly depolymerized actin filaments, while the subsequent addition of monomer hapten rapidly recovered actin polymerization. Whereas cofilin, an actin-severing protein, was temporally dephosphorylated several minutes after a multivalent antigen stimulation and the addition of monomer hapten rapidly increased cofilin phosphorylation level within 30 s. The removal of extracellular Ca2+ instead of monomer hapten addition did not restore cofilin phosphorylation, suggesting that the significant decrease in [Ca2+]i by monovalent hapten was not a critical reason for the actin rearrangement. Additionally, monovalent hapten did not completely reduce [Ca2+]i in mast cells pretreated with jasplakinolide, an inhibitor of actin depolymerization. These results suggest that the multivalent antigen-induced actin depolymerization mediated by cofilin dephosphorylation, and the subsequent addition of monovalent hapten in the F-actin severing state efficiently elicited actin re-polymerization by cofilin phosphorylation.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Mastocitos/metabolismo , Receptores de IgE/metabolismo , Animales , Señalización del Calcio , Línea Celular , Citocalasina D/farmacología , Mastocitos/efectos de los fármacos , Ovalbúmina/farmacología , Faloidina/química , Faloidina/metabolismo , Fosforilación , Polimerizacion , Ratas , Rodaminas/química , Rodaminas/metabolismoRESUMEN
We previously showed that cationic liposomes composed of cholesteryl-3ß-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) inhibited mast cell degranulation mediated by the cross-linking of high-affinity IgE receptors (FcεRI). In this study, we prepared three kinds of cationic liposomes composed of OH-Chol and DOPE in different ratios (0.28, 0.60, and 0.86 of OH-Chol in mol ratio, named as L-liposome, M-liposome, and H-liposome, respectively) and investigated their effects on mast cell activation. We found that mast cell degranulation evoked with antigen was inhibited by pretreatment with cationic liposomes in the composite ratio-dependent manner of OH-Chol and that the H-liposome showed the highest inhibitory effect on degranulation among three kinds of liposomes. Store-operated Ca2+ entry, phosphorylation of PI3K and Akt, and IL-4 secretion after antigen stimulation were reduced in dose-dependent manner of each liposome, but there were no differences between H-liposome and M-liposome. Meanwhile, microtubule acetylation, which is involved in the secretory granule transport, was significantly suppressed by H-liposome compared with M-liposome. These data suggested that the lipid composition in cationic liposomes themselves largely influenced the inhibition of mast cell activation as well as the efficiency of gene transfection.
Asunto(s)
Lípidos/farmacología , Mastocitos/efectos de los fármacos , Animales , Calcio/metabolismo , Cationes/química , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Lípidos/química , Liposomas/química , Mastocitos/metabolismo , RatasRESUMEN
Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross-linking of high-affinity immunoglobulin E receptor in a time- and dose-dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+ ]i ) via the inhibition of store-operated Ca2+ entry. Further, we revealed that the mechanism underlying an impaired [Ca2+ ]i increase is the inhibition of the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Yet, how cationic liposomes inhibit the PI3K-Akt pathway is still unclear. Here, we focused on caveolin-1, a major component of caveolae, which is reported to be involved in the activation of the PI3K-Akt pathway in various cell lines. In this study, we showed that caveolin-1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin-1 to the plasma membrane and the colocalization of caveolin-1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.
Asunto(s)
Calcio/metabolismo , Caveolas/metabolismo , Caveolina 1/metabolismo , Liposomas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Mastocitos/citología , Mastocitos/metabolismo , RatasRESUMEN
Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Haptenos/inmunología , Inmunoglobulina G/inmunología , Mastocitos/inmunología , Animales , Línea Celular Tumoral , Recubrimiento Inmunológico/inmunología , Mastocitos/citología , Ratones , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/inmunología , Ratas , Receptores de IgE/inmunología , Receptores de IgG/inmunologíaRESUMEN
Mast cells (MCs) are important effectors of the immediate allergic response. MCs are distributed throughout various tissues and organs, and adhere to extracellular matrix (ECM) with broad stiffness in the body. Here we compared cellular responses following antigen stimulation in MCs on glass-base dishes with and without a hydrogel. We found that an antigen-induced increase in intracellular Ca2+ concentration was suppressed slightly in cells on hydrogel-coated dishes compared with those on non-coated dishes, whereas their subsequent degranulation was largely inhibited in cells adherent to the hydrogel. Focusing on focal adhesions (FAs), vinculin was distributed in a dot-like manner at the bottom of resting cells on non-coated dishes but not on hydrogel-coated dishes. According to antigen stimulation, phosphorylation of focal adhesion kinase and additive vinculin accumulation to FAs were promoted in cells on non-coated dishes, but were diminished on hydrogel-coated dishes. Moreover, microtubule reorganization and acetylation (which have important roles in MC degranulation) were also suppressed in activated MCs adherent to the hydrogel. These findings suggest that adhesion to a hydrogel led to failure of composition of functional FAs and microtubule tracts, which resulted in suppression of MC degranulation following antigen stimulation.
Asunto(s)
Degranulación de la Célula , Hidrogeles/farmacología , Mastocitos/fisiología , Microtúbulos/efectos de los fármacos , Microtúbulos/patología , Andamios del Tejido/química , Animales , Calcio/metabolismo , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/fisiología , Línea Celular , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Hidrogeles/química , Mastocitos/citología , Mastocitos/efectos de los fármacos , Microtúbulos/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Propiedades de SuperficieRESUMEN
OBJECTIVE: The aim of this study was to investigate whether microtubule acetylation is triggered by antigen stimulation and how it affects mast cell degranulation. METHODS: The RBL-2H3 cell line was used as a model for mast cells. Acetylation of α-tubulin was analyzed by Western blotting. Intracellular distribution of α-tubulin and acetylated α-tubulin was observed by immunostaining. Degranulation was monitored by measuring the activity of ß-hexosaminidase secreted into cell supernatants. Tukey-Kramer test was used to compare differences between groups. RESULTS: Microtubule acetylation proceeds globally in mast cell cytoplasm after antigen stimulation in addition to accelerated formation of microtubule-organizing centers. Pretreatment with 5Z-7-oxozeaenol (5 µmol/l), an inhibitor of TGF-ß-activated kinase 1, which is a key activator of α-tubulin acetyltransferase 1, did not affect the distribution and acetylation of microtubules in resting cells; however, it significantly suppressed antigen-evoked microtubule acetylation and their reorganization, and subsequent degranulation (95.0 ± 1.2% inhibition, n = 3, P < 0.01). CONCLUSIONS: These results provided new insight into the post-translational modifications of microtubule to regulate mast cell degranulation.
Asunto(s)
Antígenos/fisiología , Mastocitos/fisiología , Microtúbulos/fisiología , Tubulina (Proteína)/fisiología , Acetilación , Animales , Degranulación de la Célula , Línea Celular , Ratas , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.
RESUMEN
Autonomic neurons innervate pancreatic islets of Langerhans and participate in the maintenance of blood glucose concentrations by controlling hormone levels through attachment with islet cells. We previously found that stimulated superior cervical ganglia (SCG) could induce Ca2+ oscillation in α cells via neuropeptide substance P using an in vitro co-culture model. In this study, we studied the effect of SCG neurite adhesion on intracellular secretory granule movement and glucagon secretion in α cells stimulated by low glucose concentration. Spinning disk microscopic analysis revealed that the mean velocity of intracellular granules was significantly lower in α cells attached to SCG neurites than that in those without neurites under low (2 mM), middle (10 mM), and high (20 mM) glucose concentrations. Stimulation by a low (2 mM) glucose concentration significantly increased glucagon secretion in α cells lacking neurites but not in those bound to neurites. These results suggest that adhesion to SCG neurites decreases low glucose-induced glucagon secretion in pancreatic α cells by attenuating intracellular granule movement activity.
Asunto(s)
Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Neuritas/metabolismo , Vesículas Secretoras/metabolismo , Ganglio Cervical Superior/metabolismo , Animales , Línea Celular , Técnicas de Cocultivo , Células Secretoras de Glucagón/citología , Ratones , Ratones Endogámicos BALB C , Ganglio Cervical Superior/citologíaRESUMEN
Cationic liposomes are commonly used as vectors to effectively introduce foreign genes (antisense DNA, plasmid DNA, siRNA, etc.) into target cells. Cationic liposomes are also known to affect cellular immunocompetences such as the mast cell function in allergic reactions. In particular, we previously showed that the cationic liposomes bound to the mast cell surface suppress the degranulation induced by cross-linking of high affinity IgE receptors in a time- and dose-dependent manner. This suppression is mediated by impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+]i) via inhibition of store-operated Ca2+ entry (SOCE). Here we study the mechanism underlying an impaired [Ca2+]i increase by cationic liposomes in mast cells. We show that cationic liposomes inhibit the phosphorylation of Akt and PI3 kinases but not Syk and LAT. As a consequence, SOCE is suppressed but Ca2+ release from endoplasmic reticulum (ER) is not. Cationic liposomes inhibit the formation of STIM1 puncta, which is essential to SOCE by interacting with Orai1 following the Ca2+ concentration decrease in the ER. These data suggest that cationic liposomes suppress SOCE by inhibiting the phosphorylation of PI3 and Akt kinases in mast cells.
Asunto(s)
Calcio/metabolismo , Liposomas/farmacología , Mastocitos/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Señalización del Calcio , Cationes , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/análogos & derivados , Colesterol/química , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Transporte Iónico/efectos de los fármacos , Liposomas/química , Mastocitos/citología , Mastocitos/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Fosfatidiletanolaminas/química , Fosfatidilinositol 3-Quinasa/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Cationic liposomes have attracted recent attention as DNA vaccine carriers that can target dendritic cells (DCs). In general, cationic liposome/DNA complexes (lipoplexes) are taken up by various cells via clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or phagocytosis, with the mode of endocytosis determining further intracellular trafficking pathways. Moreover, the physicochemical properties of cationic lipoplexes, including lipid composition, shape, size, and charge, influence transfection efficiency, affecting uptake and subsequent intracellular pathways. To develop cationic liposomes as potential DNA vaccine carriers, the objective of this study was to study the effect of lipoplex size on DNA transfection efficiency in DCs. We explored the size-dependent endocytosis pathway and the intracellular trafficking of cationic lipoplexes using bone marrow derived dendritic cells (BMDCs). Our results indicated that small-sized lipoplexes (approximately 270nm diameter) were taken up by BMDCs via caveolae-mediated endocytosis, which led to a non-degradative pathway, whereas larger-sized lipoplexes (approximately 500nm diameter) were taken up by BMDCs via clathrin-mediated endocytosis and micropinocytosis, which led to a lysosomal degradation pathway. These findings suggest that, by regulating the size of lipoplexes, it may be possible to develop cationic liposomes as DNA vaccine therapies for targeting DCs.
Asunto(s)
ADN/administración & dosificación , ADN/química , Células Dendríticas/metabolismo , Transfección/métodos , Liposomas , Luciferasas/genética , Luciferasas/metabolismo , PlásmidosRESUMEN
We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.
Asunto(s)
Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Mediciones Luminiscentes/métodos , Microscopía por Video/métodos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Línea Celular , Copépodos/enzimología , Células Secretoras de Glucagón/efectos de los fármacos , Glucosa/farmacología , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Microscopía Confocal , Cloruro de Potasio/farmacología , Proglucagón/genética , Proglucagón/metabolismo , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismoRESUMEN
Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 µm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 µm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Secretoras de Glucagón/metabolismo , Inmunoglobulinas/metabolismo , Animales , Western Blotting , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Citoesqueleto/metabolismo , Exocitosis/efectos de los fármacos , Células Secretoras de Glucagón/efectos de los fármacos , Inmunoglobulinas/genética , Ratones , Microscopía Confocal , Microscopía por Video , Nocodazol/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.
Asunto(s)
Mastocitos/metabolismo , Proteínas Qa-SNARE/metabolismo , Animales , Células Cultivadas , Exocitosis , Fosforilación , Unión Proteica , Ratas , Proteínas SNARE/metabolismo , Treonina/metabolismoRESUMEN
Calcium ion (Ca(2+)) uptake into the mitochondrial matrix influences ATP production, Ca(2+) homeostasis, and apoptosis regulation. Ca(2+) uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca(2+) uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca(2+)-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca(2+) entry into the matrix. Mitochondrial Ca(2+) uptake 1 (MICU1) functions as a Ca(2+)-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca(2+) in two steps. Consequently, mitochondrial Ca(2+) uptake likely plays a role in cellular function through cytosolic Ca(2+) buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca(2+) uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca(2+) elevation after antigen stimulation. Moreover, ß-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca(2+) uptake in mast cells, while MCU plays a role in mast cell degranulation.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Degranulación de la Célula , Mastocitos/metabolismo , Mitocondrias/metabolismo , Animales , Antígenos/inmunología , Canales de Calcio/genética , Línea Celular , Proliferación Celular , Dinitrofenoles/inmunología , Regulación de la Expresión Génica , Mastocitos/inmunología , Potencial de la Membrana Mitocondrial , Mitocondrias/inmunología , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Interferencia de ARN , Ratas , Albúmina Sérica Bovina/inmunología , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Recent studies have revealed that SNARE proteins are involved in exocytotic release in mast cells. Previously, we reported that mast cell SNARE proteins induce membrane fusion between liposomes. Moreover, we found that synaptotagmin 2, a candidate Ca2+ sensor for mast cell exocytosis, enhanced SNARE-mediated membrane fusion via Ca2+ and phosphatidylserine. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an acidic phospholipid like phosphatidylserine. In the present study, we investigated whether PIP2 is involved in the enhancement effect of synaptotagmin 2 on SNARE-mediated membrane fusion. PIP2 did not show any significant effect on SNARE-mediated membrane fusion by itself. In the presence of Ca2+, synaptotagmin 2 enhanced SNARE-mediated membrane fusion between liposomes containing PIP2. However, even in the presence of Ca2+, when we used 100% PC liposomes, synaptotagmin 2 did not show any significant effect on SNARE-mediated membrane fusion. These results indicated that PIP2 is involved in the enhancement effect of synaptotagmin 2 on membrane fusion between liposomes containing mast cell SNARE proteins.
Asunto(s)
Liposomas/química , Proteínas de la Fusión de la Membrana/química , Fusión de Membrana , Fosfatidilinositol 4,5-Difosfato/química , Proteínas SNARE/química , Sinaptotagmina II/químicaRESUMEN
Nano vectors are useful tools to deliver foreign DNAs, oligonucleotides, and small interfering double-stranded RNAs (siRNAs) into mammalian cells with gene transfection and gene regulation. In such experiments we have found the liposomes with a biosurfacant mannosylerythriol lipid (MEL-A) are useful because of their high transfer efficiency, and their unique mechanism to transfer genes to target cells with the lowest toxicity. In the present review we will describe our current work, which may contribute to the great advance of gene transfer to target cells and gene regulations. For more than two decades, the liposome technologies have changed dramatically and various methods have been proposed in the fields of biochemistry, cell biology, biotechnology, and so on. In addition, they were towards to pharmaceutics and clinical applications. The liposome technologies were expected to use gene therapy, however, they have not reached a requested goal as of yet. In the present paper we would like to present an approach using a biosurfactant, MEL-A, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates and increases efficiency in gene transfection. The present work shows new transfection agents based on liposomes containing biosurfactant MEL-A.
RESUMEN
Several studies have shown that cationic liposomes exert immunomodulatory effects with low immunogenicity and toxicity, and offer advantages such as easy preparation and targeting. Cationic liposomes not only transport DNA to immune cells but also enhance the function of antigen presenting cells such as dendritic cells and macrophages. Here, we investigated the effect of a particular cationic liposome on mast cell function during allergic reaction. We found that the cationic liposomes bound to the mast cell surface suppressed degranulation induced by cross-linking of high affinity immunoglobulin E receptors in a time- and dose-dependent manner. The suppression of degranulation was mediated by impairment of the sustained level of intracellular Ca(2+) concentration ([Ca(2+)]i) derived from the inhibition of store-operated Ca(2+) entry. The decrease in sustained elevation of [Ca(2+)]i led to the suppression of phosphorylation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins such as SNAP-23, syntaxin-4, which are necessary for membrane fusion between secretory granules and the plasma membrane during degranulation. Furthermore, the cationic liposomes suppressed vascular permeability elevation induced by mast cell activation in mice. These results showed that cationic liposomes possess the novel property of inhibiting mast cell activation, suggesting the possibility of developing cationic liposomes as anti-allergic effectors.
Asunto(s)
Cationes , Hipersensibilidad/tratamiento farmacológico , Liposomas , Mastocitos/inmunología , Animales , Calcio/metabolismo , Permeabilidad Capilar , Degranulación de la Célula , Línea Celular , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Mastocitos/metabolismo , Fosforilación , RatasRESUMEN
Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca(2+) concentration ([Ca(2+)]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6(siRNA-CADM1) cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6(siRNA-CADM1) cells, the responding rate during 3min after SCG nerve stimulation significantly increased compared with that within 1min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6(siRNA-CADM1) cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve-α cell interaction. Pretreatment with CP99,994 (0.1µg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Células Secretoras de Glucagón/fisiología , Inmunoglobulinas/fisiología , Receptores de Neuroquinina-1/fisiología , Sustancia P/fisiología , Ganglio Cervical Superior/fisiología , Animales , Calcio/metabolismo , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Comunicación Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Inmunoglobulinas/biosíntesis , Ratones , Receptores de Neuroquinina-1/biosíntesis , Venenos de Escorpión/farmacología , Ganglio Cervical Superior/efectos de los fármacosRESUMEN
Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency.