共查询到20条相似文献,搜索用时 15 毫秒
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Lee Hedden Cyril H. Benes Stephen P. Soltoff 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011
Background
The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca2+]i. We report that some agents that can block P2X7R receptors also promote diverse P2X7R-independent effects on cell signaling.Methods
We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X7R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.Results
With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated 45Ca2+ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X7R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.Conclusions
Several agents used as P2X7R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.General significance
Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins. 相似文献4.
Christian C. Colín-Santana S. Eréndira Avendaño-VázquezRocío Alcántara-Hernández J. Adolfo García-Sáinz 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011
Background
Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA1 and EGF receptors.Methods
C9 cells expressing LPA1 receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA1 and EGF receptor phosphorylation.Results
EGF diminished LPA-mediated intracellular calcium response and induced LPA1 receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA1 receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA1 receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events.Conclusions
EGF receptors and LPA1 receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA1 receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA1 receptor function in inhibitory fashion.General significance
EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications. 相似文献5.
U. Fontanils M. Seil S. Pochet M. El Ouaaliti M. Garcia-Marcos J.P. Dehaye A. Marino 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Agonists of P2X7 receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Δψm) in exocrine glands.Methods
The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Δψm was estimated with tetramethylrhodamine.Results
Activation of P2X7 receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Δψm by ATP.Conclusions
We conclude that, in rat submandibular glands, P2X7 receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists.General significance
Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2. 相似文献6.
Yasuhiro Ohshima Mitsutoshi Tsukimoto Takato Takenouchi Hitoshi Harada Akina Suzuki Mitsuru Sato Hiroshi Kitani Shuji Kojima 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.Methods
Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X7 receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X7 receptor.Results
Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X7 receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X7 receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X7 receptor channel plays a significant role in mediating the ATP release.General Significance
We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation. 相似文献7.
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Binggang Xiang Guoying Zhang Hongmei Ren Manjula Sunkara Andrew J. Morris T. Kent Gartner Susan S. Smyth Zhenyu Li 《PloS one》2012,7(12)
Background
ADP is an important physiological agonist that induces integrin activation and platelet aggregation through its receptors P2Y1 (Gαq-coupled) and P2Y12 (Gαi-coupled). P2Y12 plays a critical role in platelet activation and thrombosis. Adenosine-based P2Y12 antagonists, 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2MeSAMP) and Cangrelor (AR-C69931MX) have been widely used to demonstrate the role of P2Y12 in platelet function. Cangrelor is being evaluated in clinical trials of thrombotic diseases. However, a recent study reported that both 2MeSAMP and Cangrelor raise intra-platelet cAMP levels and inhibit platelet aggregation through a P2Y12-independent mechanism.Methodology/Principal Findings
The present work, using P2Y12 deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and Cangrelor inhibit platelet activation and thrombosis. 2MeSAMP and Cangrelor inhibited aggregation and ATP release of wild-type but not P2Y12 deficient platelets. 2MeSAMP and Cangrelor neither raised intracellular cAMP concentrations nor induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in washed human or mouse platelets. Furthermore, unlike the activators (PGI2 and forskolin) of the cAMP pathway, 2MeSAMP and Cangrelor failed to inhibit Ca2+ mobilization, Akt phosphorylation, and Rap1b activation in P2Y12 deficient platelets. Importantly, while injection of Cangrelor inhibited thrombus formation in a FeCl3-induced thrombosis model in wild-type mice, it failed to affect thrombus formation in P2Y12 deficient mice.Conclusions
These data together demonstrate that 2MeSAMP and Cangrelor inhibit platelet function through the P2Y12-dependent mechanism both in vitro and in vivo. 相似文献9.
Andrew W. Farrell Safina GadeockAleta Pupovac Bin WangIman Jalilian Marie RansonRonald Sluyter 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor.Methods
Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA.Results
RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~ 116 μM, and this uptake was reduced in the presence of extracellular Ca2+ and Mg2+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake.Conclusions
P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.General significance
RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes. 相似文献10.
Li-xia Fan Chang-mei Liu An-hui Gao Yu-bo Zhou Jia Li 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Targeting multiple aspects of cellular metabolism, such as both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), has the potential to improve cancer therapeutics. Berberine (BBR), a widely used traditional Chinese medicine, exerts its antitumor effects by inhibiting OXPHOS. 2-Deoxy-d-glucose (2-DG) targets aerobic glycolysis and demonstrates potential anticancer effects in the clinic. We hypothesized that BBR in combination with 2-DG would be more efficient than either agent alone against cancer cell growth.Methods
The effects of BBR and 2-DG on cancer cell growth were evaluated using the Sulforhodamine B (SRB) method. Cell death was detected with the PI uptake assay, and Western blot, Q-PCR and luciferase reporter assays were used for signaling pathway detection. An adenovirus system was used for gene overexpression.Results
BBR combined with 2-DG synergistically enhanced the growth inhibition of cancer cells in vitro. Further mechanistic studies showed that the combination drastically enhanced ATP depletion and strongly disrupted the unfolded protein response (UPR). Overexpressing GRP78 partially prevented the cancer cell inhibition induced by both compounds.Conclusions
Here, we report for the first time that BBR and 2-DG have a synergistic effect on cancer cell growth inhibition related to ATP energy depletion and disruption of UPR.General significance
Our results propose the potential use of BBR and 2-DG in combination as an anticancer treatment, reinforcing the hypothesis that targeting both aerobic glycolysis and OXPHOS provides more effective cancer therapy and highlighting the important role of UPR in the process. 相似文献11.
This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y2 receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP3 receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca2+-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y2 receptors. These results show that P2Y2 receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP3/intracellular Ca2+ release and Ca2+ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases. 相似文献
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Maria-Ioanna Ellina Panagiotis Bouris Alexios J. Aletras Achilleas D. Theocharis Dimitris Kletsas Nikos K. Karamanos 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.Methods
Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.Results
EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.Conclusions and general significance
The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. 相似文献13.
Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic
nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated
kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway
involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate
(UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating
kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the
most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent
agonists, characteristic of the P2Y2 or P2Y4 receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The
receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially
inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2
phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor
GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is
mediated by a P2Y2 or P2Y4 receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR. 相似文献
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Ilya V. Fedorov 《生物化学与生物物理学报:生物膜》2007,1768(7):1727-1740
Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca2+ mobilization. Based on a mathematical model of purinergic Ca2+ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y2 and P2Y4 receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca2+. Cellular responses versus concentration of BzATP, a P2Y2 agonist and a P2Y4 antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y2 and P2Y11 are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y2/P2Y4 homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y2 and P2Y4 receptors operative mostly in the dimeric form. 相似文献
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Yanghui Xing Yan Gu James J. Bresnahan Emmanuel M. Paul Henry J. Donahue Jun You 《PloS one》2014,9(9)
We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow), and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice. 相似文献
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Rui Xie Jingyu Xu Guorong Wen Hai Jin Xuemei Liu Yuan Yang Bei Ji Yixia Jiang Penghong Song Hui Dong Biguang Tuo 《The Journal of biological chemistry》2014,289(27):19137-19149
ATP is an abundant biochemical component of the tumor microenvironment and a physiologic ligand for the P2Y2 nucleotide receptor (P2Y2R). In this study, we investigated the effect of ATP on the cellular behavior of human hepatocellular carcinoma (HCC) cells and the role of P2Y2R in ATP action and aimed to find a new therapeutic target against HCC. The experiments were performed in native isolated human HCC cells, normal hepatocytes, human HCC cell lines, and nude mice. We found that the mRNA and protein expression levels of P2Y2R in native human HCC cells and the human HCC cell lines HepG2 and BEL-7404 were enhanced markedly compared with human normal hepatocytes and the normal hepatocyte line LO2, respectively. ATP induced intracellular Ca2+ increases in HCC cells and promoted the proliferation and migration of HCC cells and the growth of HCC in nude mice. The P2Y receptor antagonist suramin, P2Y2R-specific shRNA, the store-operated calcium channel inhibitors 2-aminoethoxydiphenyl borate (2-APB) and 1-(β-3-(4-methoxy-phenyl) propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride (), and stromal interaction molecule (STIM1)-specific shRNA inhibited the action of ATP on HCC cells. In conclusion, P2Y2R mediated the action of ATP on the cellular behavior of HCC cells through store-operated calcium channel-mediated Ca2+ signaling, and targeting P2Y2R may be a promising therapeutic strategy against human HCC. SKF96365相似文献
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Carolina O. Souza Giani F. Santoro Vanessa R. Figliuolo Hayandra Ferreira Nanini Heitor S.P. de Souza Morgana Teixeira Lima Castelo-Branco Alessandra Alves Abalo Mauricio M. Paiva Claudia M.L.M. Coutinho Robson Coutinho-Silva 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP.Methods
We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique.Results
ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells.General significance
The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions. 相似文献18.
Aim
Copper deficiency could cause fatal hematological and neurological disorders or other diseases. Amino acids are involved in the absorption of copper ions. The purpose of this study is to evaluate the absorption of copper in amino acid complex forms and determine its mechanism in the Caco-2 cell culture model.Main methods
The human colonic adenocarcinoma cell line Caco-2 culture model was used to determine the permeability of copper ions in inorganic form (CuSO4) and the amino acid complex forms. Lysine and methionine, as well as carboplatin were used to determine the possible involvement of amino acid transporters or copper transporter 1 (CTR1).Key findings
The results showed that all of the amino acid complex forms facilitated copper absorption. The apparent permeabilities of copper ions in these complex forms were at least 7.6 fold higher than those in the CuSO4 form. The permeability rank order of copper in various amino acid complex forms was Cu-glutamate < Cu-lysine = Cu-aspartic acid = Cu methionine < Cu-arginine < Cu-(lysine/glutamate). Mechanistic studies revealed that the enhanced absorption of copper in copper amino acid complexes could be the result of enhanced uptake (as in Cu-methionine complex) or enhanced basolateral efflux (as in Cu-lysine complex). Copper transporter 1 (or CTR1) inhibitor carboplatin did not affect the absorption of copper in Cu-methionine complex, suggesting that the dominant pathway for copper amino acid complexes is not CTR1.Significance
Enhanced absorption of copper ions in amino acid complex appears to be mediated by amino acid transporters. 相似文献19.
Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP 总被引:1,自引:0,他引:1
Velázquez B Garrad RC Weisman GA González FA 《Molecular and cellular biochemistry》2000,206(1-2):75-89
The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y2 receptors. While ATP and UTP activated the murine and human P2Y2 receptors with similar potencies (EC50 values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC50 = 9.1-21.2 M) than UTP (IC50 = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization. 相似文献
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