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1.
The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the ‘low affinity’ IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium+ uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself. 相似文献
2.
Safina Gadeock Jimmy N.S.N. Tran Jennifer G. Georgiou Iman Jalilian James S. Wiley Ronald Sluyter 《生物化学与生物物理学报:生物膜》2010,1798(11):2058-2066
The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium+ uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium+ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium+ uptake in a concentration-dependent fashion with a maximum effect at 5 ng/ml and with an IC50 of ~ 0.4 ng/ml. Moreover, ATP-induced YO-PRO-12+ uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity. 相似文献
3.
The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium+ uptake into KG-l cells suspended in sucrose medium (EC50 of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium+ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC50 of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC50 of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium+ uptake into KG-1 cells. ATP-induced ethidium+ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium+ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-12+ and propidium2+ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells. 相似文献
4.
Patrick Constantinescu Kati Kovacevic Giel J.C.G.M. Bosman Ronald Sluyter 《生物化学与生物物理学报:生物膜》2010,1798(9):1797-1804
Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium+ uptake into MEL cells in a concentration-dependent fashion and with an EC50 of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium+ uptake. ATP-induced ethidium+ and YO-PRO-12+ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo. 相似文献
5.
Ryan O. Stevenson Rosanne M. Taylor James S. Wiley Ronald Sluyter 《Purinergic signalling》2009,5(3):385-394
We previously demonstrated that canine erythrocytes express the P2X7 receptor, and that the function and expression of this receptor is greatly increased compared with human erythrocytes. Using
86Rb+ (K+) and organic cation flux measurements, we further compared P2X7 in erythrocytes and mononuclear leukocytes from these species. Concentration response curves of BzATP- and ATP-induced 86Rb+ efflux demonstrated that canine P2X7 was less sensitive to inhibition by extracellular Na+ ions compared to human P2X7. In contrast, canine and human P2X7 showed a similar sensitivity to the P2X7 antagonists KN-62 and Mg2+. KN-62 and Mg2+ also inhibited ATP-induced choline+ uptake into canine and human erythrocytes. BzATP and ATP but not ADP or NAD induced ethidium+ uptake into canine monocytes, T- and B-cells. ATP-induced ethidium+ uptake was twofold greater in canine T-cells compared to canine B-cells and monocytes. KN-62 inhibited the ATP-induced ethidium+ uptake in each cell type. P2X7-mediated uptake of organic cations was 40- and fivefold greater in canine erythrocytes and lymphocytes (T- and B-cells),
respectively, compared to equivalent human cell types. In contrast, P2X7 function was threefold lower in canine monocytes compared to human monocytes. Thus, P2X7 activation can induce the uptake of organic cations into canine erythrocytes and mononuclear leukocytes, but the relative
levels of P2X7 function differ to that of equivalent human cell types. 相似文献
6.
Rachael Bartlett Leanne Stokes Stephen J. Curtis Belinda L. Curtis 《Nucleosides, nucleotides & nucleic acids》2017,36(12):736-744
The current study aimed to determine if probenecid could directly impair the canine P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate (ATP). Patch clamp measurements demonstrated that probenecid impairs ATP-induced inward currents in HEK-293 cells expressing canine P2X7. Flow cytometric measurements of ethidium+ uptake into HEK-293 cells expressing canine P2X7 showed that probenecid impairs ATP-induced pore formation in a concentration-dependent manner, with a half maximal inhibitory concentration of 158 µM. Finally, ELISA measurements revealed that probenecid impairs ATP-induced interleukin-1β release in dog blood. In conclusion, this study reveals that probenecid can directly impair canine P2X7 activation. 相似文献
7.
Guadalupe Martel-Gallegos Griselda Casas-Pruneda Filiberta Ortega-Ortega Sergio Sánchez-Armass Jesús Alberto Olivares-Reyes Becky Diebold Patricia Pérez-Cornejo Jorge Arreola 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.Methods
J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.Results
ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.Conclusions
Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.General significance
ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections. 相似文献8.
Kenji F Shoji Pablo J Sáez Paloma A Harcha Hector L Aguila Juan C Sáez 《Channels (Austin, Tex.)》2014,8(2):142-156
Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X7 receptors (P2X7Rs). However, a link between P2X7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1−/− mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1−/− mice, in which levels of P2X7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP. A23187相似文献
9.
Hiroshi Tsujikawa Takehiro Shoda Toshiyuki Mizota Kazuhiko Fukuda 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Morphine has been shown to affect the function of immune system, but the precise mechanism remains to be elucidated. The present study was aimed to clarify the mechanism for the morphine-induced immune suppression by analyzing the direct effect of morphine on human CD3+ T cells.Methods
To identify genes up-regulated by action of morphine on the opioid receptor expressed in CD3+ T cells, PCR-select cDNA subtraction was performed by the use of total RNA from human CD3+ T cells treated with morphine in the presence and absence of naloxone.Results
We show that p53 and damage-specific DNA binding protein 2 (ddb2) genes are up-regulated by morphine in a naloxone-sensitive manner. Furthermore, the results indicate that DNA damage, quantified by apurinic–apyrimidinic site counting assay and phosphorylation of Ser-15 in P53 protein, is induced in CD3+ T cells by morphine in a naloxone-sensitive manner.General significance
Because it was shown that only the κ opioid receptor gene is expressed in CD3+ T cells in the opioid receptor family, the present study suggests that morphine induces DNA damage through the action on the κ opioid receptor, which leads to immune suppression by activation of P53-mediated signal transduction. 相似文献10.
C.F. Dick A.L.A. Dos-Santos D. Majerowicz L.S. Paes N.L. Giarola K.C. Gondim A. Vieyra J.R. Meyer-Fernandes 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi.Methods
32Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na+, H+ and K+ fluxes were also investigated. The transport capacities of different evolutive forms were compared.Results
Epimastigotes grew significantly more slowly in 2 mM than in 50 mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na+. We found that the parasites express TcPho84, a H+:Pi-symporter, and TcPho89, a Na+:Pi-symporter. Both Pi influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na+-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K+ ionophore) or SCH28028 (inhibitor of (H+ + K+)ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H+ gradient energizes uphill Pi entry and that K+ recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, decreased only the Na+-dependent Pi uptake, indicating that this Na+ pump generates the Na+ gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently.Conclusions
Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na+ or H+/K+ fluxes.General significance
This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms. 相似文献11.
James P. Barnett Claudia A. Blindauer Omar Kassaar Siavash Khazaipoul Esther M. Martin Peter J. Sadler Alan J. Stewart 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another.Scope of review
Here we review the Zn2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn2 +, its effect upon cellular Zn2 + uptake and its importance in the diagnosis of myocardial ischemia are considered.Major conclusions
We previously identified the major binding site for Zn2 + on albumin. Furthermore, we revealed that Zn2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn2 +-binding to albumin in blood plasma.General significance
Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn2 + to albumin is important for the control of circulatory/cellular Zn2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin. 相似文献12.
Background
Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.Methods
Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.Results
A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).Conclusion
With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen. 相似文献13.
Jae Hong Park Jung Min Ryu Seung Pil Yun Mi Ok Kim Ho Jae Han 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
Extracellular matrix (ECM) components and intracellular pH (pHi) may serve as regulators of cell migration in various cell types.Methods
The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na+/H+ exchanger (NHE)-1 activity was evaluated by measuring pHi and [22Na+] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.Results
ECM components (FN, laminin, fibrinogen, and collagen type I) increased [22Na+] uptake, pHi, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [22Na+] uptake and pHi. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.Conclusions
FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca2 +/CaM signaling pathways.General significance
The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways. 相似文献14.
Phuong Dao-Ung Kristen K. Skarratt Stephen J. Fuller Leanne Stokes 《Purinergic signalling》2015,11(4):481-490
P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC50 of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC50 of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14+ monocytes was suppressed with trifluoperazine and paroxetine. 相似文献
15.
Shemon AN Sluyter R Stokes L Manley PW Wiley JS 《Biochemical and biophysical research communications》2008,365(3):515-520
A panel of 18 protein tyrosine kinase antagonists were tested for their inhibitory effect on human P2X7 receptor-mediated 86Rb+ (K+) efflux. The most potent compound (compound P), a phthalazinamine derivative and an inhibitor of vascular endothelial growth factor receptor kinase, blocked ATP-induced 86Rb+-efflux in human B-lymphocytes and erythrocytes by 76% and 66%, respectively. This inhibition was dose-dependent in both cell types with an IC50 of ∼5 μM. Kinetic analysis showed compound P was a non-competitive inhibitor of P2X7. This compound also inhibited ATP-induced ethidium+ influx into B-lymphocytes and P2X7-transfected-HEK-293 cells, as well as ATP-induced 86Rb+-efflux from canine erythrocytes. Externally, but not internally, applied compound P impaired ATP-induced inward currents in P2X7-transfected-HEK-293 cells. This study demonstrates that a novel protein tyrosine kinase antagonist directly impairs native and recombinant human P2X7 receptors. The data suggests that antagonists which target ATP-binding sites of kinases may potentially block the P2X7 receptor. 相似文献
16.
Tae-Ho Kim Gun-Dong KimHyun-Jong Ahn Jeong-Je ChoYong Seek Park Cheung-Seog Park 《Life sciences》2013
Aims
Atopic dermatitis (AD) is a chronic and relapsing inflammatory dermatitis characterized by pruritic and eczematous skin lesions. Here, we investigated the therapeutic effect of the fruit flavonoid naringenin on DNFB induced atopic dermatitis mice model.Main methods
AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of the fruit flavonoid naringenin were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4+T cells.Key findings
Intraperitoneal injection of naringenin for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, naringenin significantly suppressed production of interferon-gamma (IFN-γ) by activated CD4+ T cells and serum IgE level. Furthermore, naringenin reduced DNFB-induced infiltration of eosinophils, mast cells, CD4+ T cells, and CD8+ T cells in skin lesions.Significance
Naringenin may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IFN-γ production of activated CD4+ T cells, serum IgE levels and infiltration of immune cells to skin lesion. 相似文献17.
J.C. Souza E.C Vanzela R.A. Ribeiro L.F. Rezende C.A. de Oliveira E.M. Carneiro H.C.F. Oliveira A.C. Boschero 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(4):769-775
Aims/hypothesis
Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca2 + handling in these islets.Methods
Isolated islets from both LDLR−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).Results
The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR−/− than in WT islets, paralleled by an impairment of Ca2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR−/− compared with WT islets. Removal of excess cholesterol from LDLR−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca2 + handling was also normalized in cholesterol-depleted LDLR−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.Conclusion
Abnormally high (LDLR−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca2 + handling. Normalization of cholesterol improves Ca2 + handling and insulin secretion in LDLR−/− islets. 相似文献18.
Da-En Cheng Jen-Yu Hung Ming-Shyan Huang Ya-Ling Hsu Chi-Yu Lu Eing-Mei Tsai Ming-Feng Hou Po-Lin Kuo 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.Methods
Circulating CD14+CD16+ of breast cancer patients and induced CD14+CD16+ DCs were identified as tDCs by treating CD14+ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.Results
The CD14+CD16+ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c+ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c+/Ly6G+/IL-10+ in the BALB/c mice.Conclusions
Galectin-1 can induce tDCs after internalizing into CD14+ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.General significance
Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development. 相似文献19.
20.
Alexis Nazareno Campetelli Noelia Edith Monesterolo Gabriela Previtali Verónica Silvina Santander Marina Rafaela Amaiden Carlos Angel Arce Javier Valdez-Taubas César Horacio Casale 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013