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1.
Chem Commun (Camb) ; 47(33): 9462-4, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21785757

RESUMO

A concise method for the formation of cyclopyrophosphate of cIDPRE as well as sulfur and selenium-substituted pyrophosphate cIDPRE analogues (P(1)(S)-cIDPRE, P(1)(Se)-cIDPRE, P(2)(S)-cIDPRE and P(2)(Se)-cIDPRE) was reported and one of the P(S)-diastereoisomers, P(1)(S)-cIDPRE-1, is a novel membrane-permeant cADPR antagonist.


Assuntos
ADP-Ribose Cíclica/análogos & derivados , Difosfatos/química , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Inosina Monofosfato/análogos & derivados , Inosina Monofosfato/síntese química , Inosina Monofosfato/química , Conformação Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Selênio/química , Estereoisomerismo , Enxofre/química
2.
Artigo em Alemão | MEDLINE | ID: mdl-19669708

RESUMO

Gain of basic and applied medical knowledge and the changing demands of society with regard to medical professions are the main factors for continuous reforms in medical curricula. The Bologna Declaration of 1999 initiated the development of a unified European higher education area. A key tool for unification is the introduction of the Bachelors/Masters system. Although some European countries have adapted their medical curricula to the Bachelors/Masters system there is still debate on this issue in Germany. Some societies, e.g., the Society for Medical Education, demonstrated how the Bachelors/Masters system might be transferred to Germany. Moreover, the German Association of Medical Students already published a core curriculum compatible with the Bologna criteria. Some central elements of the Bologna Declaration have already been or could easily be integrated into the current structure of medical studies, e.g., quality assurance or a credit point transfer system. Furthermore, in the framework of the German medical licensure law, it is possible to introduce a curriculum fully compatible with the Bologna Declaration. A meaningful prerequisite would be a unified national (or European) qualification frame and catalog of learning objectives, designed according to the Bologna criteria. This should guarantee good mobility for medical students within Europe.


Assuntos
Currículo/normas , Currículo/tendências , Educação de Graduação em Medicina/organização & administração , Guias como Assunto , Europa (Continente) , União Europeia , Alemanha
3.
Biochem Soc Trans ; 35(Pt 1): 109-14, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17233614

RESUMO

cADPR [cyclic ADPR (ADP-ribose)], NAADP (nicotinic acid-adenine dinucleotide phosphate) and ADPR belong to the family of adenine-containing second messengers. They are metabolically related and are all involved in the regulation of cellular Ca(2+) homoeostasis. Activation of specific plasma membrane receptors is connected to cADPR formation in many cell types and tissues. In contrast receptor-mediated formation of NAADP and ADPR has been shown only in a few selected cellular systems. The intracellular Ca(2+) channel triggered by cADPR is the RyR (ryanodine receptor); in the case of NAADP, both activation of RyR and a novel Ca(2+) channel have been proposed. In contrast, ADPR opens the non-specific cation channel TRPM2 [TRP (transient receptor potential) melastatin 2] that belongs to the TRP family of ion channels.


Assuntos
Adenina/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Animais , ADP-Ribose Cíclica/química , Humanos , Modelos Biológicos , Modelos Químicos , NADP/análogos & derivados , NADP/química , Receptores Purinérgicos P2/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais , Canais de Cátion TRPM/metabolismo
4.
Br J Pharmacol ; 149(4): 337-44, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16967053

RESUMO

BACKGROUND AND PURPOSE: Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues. EXPERIMENTAL APPROACH: cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells. KEY RESULTS: Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5'-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds. CONCLUSIONS AND IMPLICATIONS: The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , ADP-Ribose Cíclica/farmacologia , Nucleotídeos de Inosina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Antineoplásicos/metabolismo , Cálcio/metabolismo , ADP-Ribose Cíclica/análogos & derivados , ADP-Ribose Cíclica/metabolismo , Citocinas/metabolismo , Humanos , Hidrólise , Nucleotídeos de Inosina/química , Células Jurkat , NAD+ Nucleosidase/metabolismo , Linfócitos T/metabolismo
5.
Cell Signal ; 13(12): 895-9, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11728829

RESUMO

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting alpha6beta1-integrin-mediated Ca(2+) signalling. Both Ca(2+) release from thapsigargin-sensitive Ca(2+) stores and capacitative Ca(2+) entry via Ca(2+) channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of alpha6beta1-integrin-mediated Ca(2+) signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca(2+)-releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Sinalização do Cálcio , Integrinas/fisiologia , Linfócitos T/imunologia , Adenosina Difosfato Ribose/antagonistas & inibidores , Adenosina Difosfato Ribose/farmacologia , Adenosina Difosfato Ribose/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , ADP-Ribose Cíclica , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Humanos , Imidazóis/farmacologia , Integrina alfa6beta1 , Células Jurkat , Cinética , Laminina/farmacologia , Pirimidinas/farmacologia , Pirrolidinonas/farmacologia , Linfócitos T/efeitos dos fármacos , Tapsigargina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/fisiologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/fisiologia
6.
Anal Biochem ; 299(2): 218-26, 2001 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11730346

RESUMO

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Antígenos CD , Antígenos de Diferenciação/análise , Cromatografia Líquida de Alta Pressão/métodos , NAD+ Nucleosidase/análise , NAD/análogos & derivados , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/análise , Animais , Aplysia/enzimologia , Humanos , Células Jurkat , Leucemia , Glicoproteínas de Membrana , NAD/metabolismo , Frações Subcelulares/enzimologia , Linfócitos T
7.
Eur J Biochem ; 268(21): 5601-8, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11683883

RESUMO

In recent years, evidence has accumulated that NAD+ serves as a precursor of metabolites that are involved in a number of regulatory processes. In this work we show that extracellularly added NAD+ was rapidly degraded by intact human monocytes to nicotinamide and ADP-ribose. Besides these main products, minor amounts of AMP, ADP and cADP-ribose were formed. Expression of CD38, which has been identified as NAD+-glycohydrolase (EC 3.2.2.6) degrading NAD+ into nicotinamide and ADP-ribose, was determined on freshly isolated human monocytes by flow cytometry and RT-PCR. Upon ligation with anti-CD38 mAb, CD38 underwent internalization, shedding and new expression. As monocytes possess an intracellular CD38 pool, it could serve as a source for newly expressed CD38. Differentiation of monocytes to macrophages resulted in down-regulation of surface expression of CD38. This decrease correlates with a reduction in NADase activity, indicating that the amount of functional active CD38 molecules decrease during differentiation. As CD38 mRNA was found to be diminished in macrophages, regulation of the gene product seems to occur at the level of transcription or mRNA stability.


Assuntos
Antígenos CD , Antígenos de Diferenciação/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , NAD+ Nucleosidase/metabolismo , NAD/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Anticorpos Monoclonais/farmacologia , Antígenos de Diferenciação/efeitos dos fármacos , Antígenos de Diferenciação/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Regulação para Baixo , Humanos , Glicoproteínas de Membrana , Monensin/farmacologia , NAD+ Nucleosidase/efeitos dos fármacos , NAD+ Nucleosidase/imunologia , Niacinamida/metabolismo , Inibidores da Síntese de Proteínas/farmacologia
8.
J Biol Chem ; 276(37): 34722-7, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11466305

RESUMO

The ryanodine receptor of Jurkat T lymphocytes was phosphorylated on tyrosine residues upon stimulation of the cells via the T cell receptor/CD3 complex. The tyrosine phosphorylation was transient, reaching a maximum at 2 min, and rapidly declined thereafter. In co-immunoprecipitates of the ryanodine receptor, the tyrosine kinases p56(lck) and p59(fyn) were detected. However, only p59(fyn) associated with the ryanodine receptor in a stimulation-dependent fashion. Both tyrosine kinases, recombinantly expressed as glutathione S-transferase (GST) fusion proteins, phosphorylated the immunoprecipitated ryanodine receptor in vitro. In permeabilized Jurkat T cells, GST-p59(fyn), but not GST-p56(lck), GST-Grb2, or GST alone, significantly and concentration-dependently enhanced Ca(2+) release by cyclic ADP-ribose. The tyrosine kinase inhibitor PP2 specifically blocked the effect of GST-p59(fyn). This indicates that intracellular Ca(2+) release via ryanodine receptors may be modulated by tyrosine phosphorylation during T cell activation.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Linfócitos T/metabolismo , Tirosina/metabolismo , Cálcio/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-fyn
9.
Biochim Biophys Acta ; 1498(2-3): 122-33, 2000 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-11108956

RESUMO

The elevation of the cytosolic and nuclear Ca(2+) concentration is a fundamental signal transduction mechanism in almost all eukaryotic cells. Interestingly, three Ca(2+)-mobilising second messengers, D-myo-inositol 1,4,5-trisphosphate (InsP(3)), cyclic adenosine diphosphoribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP(+)) were identified in a phylogenetically wide range of different organisms. Moreover, in an as yet very limited number of cell types, sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T-lymphocytes, all three Ca(2+)-mobilising ligands have been shown to be involved in the generation of Ca(2+) signals. This situation raises the question why during evolution all three messengers have been conserved in the same cell type. From a theoretical point of view the following points may be considered: (i) redundant mechanisms ensuring intact Ca(2+) signalling even if one system does not work, (ii) the need for subcellularly localised Ca(2+) elevations to obtain a certain physiological response of the cell, and (iii) tight control of a physiological response of the cell by a temporal sequence of Ca(2+) signalling events. These theoretical considerations are compared to the current knowledge regarding the three messengers in sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T lymphocytes.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Sistemas do Segundo Mensageiro , Adenosina Difosfato Ribose/genética , Animais , Evolução Biológica , Linhagem Celular , ADP-Ribose Cíclica , Humanos , Inositol 1,4,5-Trifosfato/genética , NADP/genética , Transdução de Sinais
10.
J Cell Biol ; 150(3): 581-8, 2000 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-10931869

RESUMO

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.


Assuntos
Sinalização do Cálcio , Ativação Linfocitária , NADP/análogos & derivados , Linfócitos T/metabolismo , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/metabolismo , Complexo CD3/metabolismo , ADP-Ribose Cíclica , Relação Dose-Resposta a Droga , Humanos , Processamento de Imagem Assistida por Computador , Inositol 1,4,5-Trifosfato/metabolismo , Células Jurkat , Microinjeções , NADP/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
11.
J Mol Med (Berl) ; 78(1): 26-35, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10759027

RESUMO

The Ca2+-mobilizing natural compound cyclic ADP-ribose was discovered in sea urchin egg homogenates. Recently the involvement of cyclic ADP-ribose in Ca2+ signaling has been demonstrated in diverse biological systems spanning protozoa, plants, and cells from invertebrate, mammalian, and human sources. ADP-ribosyl cyclases synthesize cyclic ADP-ribose. Several candidate proteins for these enzymes have been proposed, including membrane-bound NAD+ glycohydrolases such as CD38 and soluble enzyme activities from various tissues and cells. Ca2+ mobilization by cyclic ADP-ribose is believed to proceed via the ryanodine receptor/Ca2+ channel, probably via binding proteins for cyclic ADP-ribose. Several antagonistic derivatives of cyclic ADP-ribose have been synthesized, some of which have been successfully used to demonstrate the involvement of cyclic ADP-ribose in sea urchin egg fertilization, glucose-dependent insulin secretion in pancreatic beta-cells, and activation and proliferation of human T-lymphocytes.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Antígenos CD , Sinalização do Cálcio/fisiologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Cálcio/metabolismo , ADP-Ribose Cíclica , Fertilização/fisiologia , Humanos , Ilhotas Pancreáticas/fisiologia , Glicoproteínas de Membrana , NAD+ Nucleosidase/metabolismo , Ouriços-do-Mar , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia
12.
Br J Pharmacol ; 128(6): 1235-40, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10578137

RESUMO

1 Recently, we provided evidence for cyclic adenosine 5'-diphosphate-ribose, cADP-ribose, as a second messenger in Jurkat T-lymphocytes upon stimulation of the T-cell receptor/CD3- complex (Guse et al., 1999). cADP-ribose mobilizes Ca2+ from an intracellular Ca2+ store which is sensitive to caffeine and gated by the ryanodine receptor/Ca2+ release channel. In the present study we investigated the ability of the trypanocidal drug, suramin, to activate the ryanodine receptor of T-cells. Since suramin cannot permeate the plasma membrane, it was necessary to microinject the drug into Fura-2 loaded T-lymphocytes. 2 In a dose dependent manner suramin increased the intracellular Ca2+ concentration. The dose-response curve is very steep and calculates for an EC50 of 7. 6+/-2.9 mM suramin in the injection pipette. 3 Co-injection of the selective ryanodine receptor inhibitor ruthenium red completely abolished the suramin induced Ca2+ transient. This finding allows for the conclusion that the IP3-receptor sensitive Ca2+ pool is not the primary target of the suramin induced Ca2+ transient. 4 Furthermore, Ins(1,4,6)PS3, an antagonist of the InsP3-receptor could not suppress the suramin-induced Ca2+ signal. The suramin induced Ca2+ transients declined very slowly; however, in the presence of Ins(1,4,6)PS3 this decay was accelerated. In addition, suramin did not interact with the cADP-ribose binding site of the ryanodine receptor of T-cells. 5 In conclusion, suramin is found to be an agonist for the T-cell ryanodine receptor as previously found for the cardiac and skeletal muscle isoform. Therefore, suramin can be designated a universal ryanodine receptor agonist.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/farmacologia , Cálcio/metabolismo , ADP-Ribose Cíclica , Relação Dose-Resposta a Droga , Humanos , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacologia , Células Jurkat , Microinjeções , Compostos Organotiofosforados/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Suramina/farmacologia , Linfócitos T/metabolismo
13.
Cell Signal ; 11(8): 611-9, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10433522

RESUMO

Interaction of Jurkat T-lymphocytes with two extracellular matrix (ECM) proteins of the basement membrane, laminin or collagen type IV, combined with poly-L-lysine resulted in a strong adhesion, a highly increased intracellular Ca2+-concentration ([Ca2]i), as compared to cells on laminin or collagen type IV alone and in spreading of the cells. The strong adhesion was independent of an increase in [Ca2+]i, was not mediated by a beta1-integrin, and was due to charge interaction between the positively charged polyaminoacid and the negatively charged cell surface. The latter was confirmed by substitution of poly-L-lysine by other positively charged polyaminoacids. In contrast, Ca+-signalling and spreading of the cells adhering to laminin or collagen type IV combined with poly-L-lysine was completely blocked by anti-beta1 mAb. However, spreading of the cells was independent of an increase in [Ca2+]i suggesting divergent signal transduction pathways leading to Ca2+-signalling and spreading of the cells. We elucidated these signal transduction pathways by inhibition of key enzymes involved. The tyrosine kinase inhibitor genistein blocked Ca2+-signalling as well as spreading, whereas inhibitors of PKC (calphostin C, GF109203x), PLCgamma (U73122) and PLA2 (bromophenacyl-bromide (BPB), 3-[4-octadecyl)benzoyl]acrylic acid (OBAA)) selectively blocked spreading of the cells.


Assuntos
Sinalização do Cálcio , Movimento Celular , Integrina beta1/metabolismo , Linfócitos T/fisiologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Colágeno/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/metabolismo , Células Jurkat , Laminina/metabolismo , Peptídeos/metabolismo , Fosfolipase C gama , Fosfolipases A/metabolismo , Fosfolipases A2 , Ácido Poliglutâmico/metabolismo , Polilisina/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tapsigargina/farmacologia , Fosfolipases Tipo C/metabolismo
14.
Cell Signal ; 11(5): 309-16, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10376802

RESUMO

Cyclic ADP-ribose (cADPR) was discovered as a potent Ca2+-mobilising natural compound in sea urchin eggs. Recently, cADPR was reported to stimulate Ca2+ signalling in several higher eukaryotic cell systems (e.g., smooth and cardiac muscle cells, neuronal cells, adrenal chromaffin cells, macrophages, pancreatic acinar cells and T-lymphocytes). The following aspects of the role of cADPR as a Ca2+-mobilising second messenger are reviewed: coupling of metabolism of cADPR to stimulation of receptors in the plasma membrane, properties and pharmacology of Ca2+ release by cADPR and the involvement of cADPR in Ca2+ entry.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Antígenos CD , Cálcio/metabolismo , Sistemas do Segundo Mensageiro , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Sinalização do Cálcio , ADP-Ribose Cíclica , Humanos , Glicoproteínas de Membrana , NAD+ Nucleosidase/metabolismo , Receptores de Superfície Celular/metabolismo
15.
Nature ; 398(6722): 70-3, 1999 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-10078531

RESUMO

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Sinalização do Cálcio , Cálcio/metabolismo , Sistemas do Segundo Mensageiro , Linfócitos T/metabolismo , Adenosina Difosfato Ribose/antagonistas & inibidores , Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/farmacologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Cromatografia Líquida de Alta Pressão , ADP-Ribose Cíclica , Ativação Enzimática , Humanos , Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Inositol 1,4,5-Trifosfato/metabolismo , Células Jurkat , Ativação Linfocitária , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Linfócitos T/imunologia
16.
FEBS Lett ; 439(3): 291-6, 1998 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-9845340

RESUMO

Cyclic ADP-ribose (cADPR) is a natural metabolite of beta-NAD+ with a potent Ca2+-mobilizing activity in different cell types, including T-lymphocytes. We investigated (i) whether stimulation of T-lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP-ribosyl cyclase and cADPR-hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca2+-mobilizing effects of this nucleotide in Jurkat and HPB.ALL T-lymphocytes. The anti-CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca2+ concentration ([Ca2+]i). In contrast, activation of an ectocellular ADP-ribosyl cyclase by preincubation of cells with beta-NAD+ led to a dose-dependent increase in cADPR, but no changes in [Ca2+]i were observed. However, extensive washing of the cells following preincubation with NAD+ demonstrated that the increases in cADPR were not intracellular but due to cell surface-associated nucleotide. Accordingly, measurements of ADP-ribosyl cyclase activity in intact T-cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP-ribosyl cyclase activity of CD38 on the regulation of the cADPR-mediated intracellular Ca2+-signalling in T-lymphocytes.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Antígenos CD , Antígenos de Diferenciação/fisiologia , Sinalização do Cálcio , NAD+ Nucleosidase/fisiologia , Linfócitos T/fisiologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/fisiologia , Antígenos de Diferenciação/metabolismo , Transporte Biológico , Western Blotting , Catálise , ADP-Ribose Cíclica , Citometria de Fluxo , Humanos , Células Jurkat , Glicoproteínas de Membrana , NAD+ Nucleosidase/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo
17.
Crit Rev Immunol ; 18(5): 419-48, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9784968

RESUMO

Ca2+ signaling in response to antigenic stimulation is essential for proliferation of T cells and therefore is one of the important early events in T-lymphocyte signal transduction. Several aspects of T cell receptor/CD3 complex stimulated Ca2+ signaling are reviewed: generation, metabolism, function, and intracellular targets of the Ca(2+)-mobilizing second messengers inositol 1,4,5-trisphosphate and cyclic ADP-ribose, the mechanism of Ca2+ entry, and the generation of Ca2+ oscillations on the single cell level. In addition, Ca2+ signaling induced by further stimuli is discussed, including other T-lymphocyte surface receptors (e.g., CD4 or beta 1-integrins, lipids, and physical stimuli).


Assuntos
Sinalização do Cálcio , Linfócitos T/metabolismo , Animais , Complexo CD3/imunologia , Complexo CD3/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Humanos , Metabolismo dos Lipídeos , Estimulação Física , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Toxinas Biológicas/metabolismo
18.
J Chromatogr B Biomed Sci Appl ; 707(1-2): 43-50, 1998 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-9613932

RESUMO

A combined two-step high-performance liquid chromatographic (HPLC) method was developed for the analysis of endogenous levels of cyclic adenosine diphosphoribose (cADPR) in cell extracts. The detection sensitivity for cADPR was about 10 pmol. Linearity of the HPLC detection system was demonstrated in the range of 10 pmol up to 2 nmol. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotides. The method was applied to the analysis of endogenous cADPR in human T cell lines. Sequential separation of perchloric acid extracts from cells on strong anion-exchange and reversed-phase ion-pair HPLC resulted in a single symmetrical peak co-eluting with standard cADPR. The identity of this endogenous material was further confirmed by its ability to be converted to ADPR upon heating the cell samples at 80 degrees C for 2 h. Recoveries of the combined perchloric acid extraction-HPLC analysis procedures were 48.3 +/- 10.2%. The determined intracellular concentrations of cADPR in quiescent Jurkat and HPB. ALL human T cells were 198 +/- 41 and 28 +/- 9 pmol/10(8) cells, respectively. In conclusion, a non-radioactive HPLC method presenting a specificity and sensitivity suitable for precise quantification of cADPR in cell extracts was developed.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/análise , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , ADP-Ribose Cíclica , Humanos , Indicadores e Reagentes , Células Jurkat , Percloratos , Reprodutibilidade dos Testes , Linfócitos T/química
19.
Biochem Biophys Res Commun ; 240(1): 146-9, 1997 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-9367900

RESUMO

Inositol 1,2,3,5,6-pentakisphosphate (Ins(1,2,3,5,6)P5) 5-phosphatase present in fetal calf thymus has been partially purified. This enzyme was inhibited dose-dependently by different thiol modifiers like N-ethylmaleimide (NEM), p-chloromercuribenzene sulfonate (PCMBS), diamide, and phenylarsine oxide (PAO). The inhibition by PCMBS and diamide was protected by preincubation with dithiothreitol (DTT) and the phosphatase substrate, Ins(1,2,3,5,6)P5. Diamide, a compound that specifically modifies vicinal thiol groups, also blocked the 5-phosphatase dose-dependently. Specificity of this blockade was proven by using dimercaptopropanol (DMP), a compound known to protect vicinal thiol groups. DMP prevented the enzyme from inhibition by diamide. These data suggest that vicinal thiols are involved in Ins(1,2,3,5,6)P5 5-phosphatase activity.


Assuntos
Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Compostos de Sulfidrila/metabolismo , Timo/enzimologia , Animais , Arsenicais/farmacologia , Sítios de Ligação , Bovinos , Inibidores Enzimáticos/farmacologia , Feto , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Especificidade por Substrato
20.
Cell Calcium ; 22(2): 91-7, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9292227

RESUMO

The pharmacological properties of the recently described antagonist for capacitative Ca2+ entry LU 52396 were investigated and compared to known Ca2+ antagonists in Jurkat T-lymphocytes. In the first set of experiments, cells were stimulated with the anti-CD3 monoclonal antibody OKT3 and, subsequently, Ca2+ antagonists were added. Under such conditions SK-F 96365, econazole, nitrendipine and ZnCl2 dose-dependently antagonized Ca2+ signaling, whereas LU 52396 in concentrations up to 100 microM did not. In contrast, when LU 52396 was added a few minutes before OKT3, a dose-dependent inhibition of the OKT3-stimulated Ca2+ signals by LU 52396 was observed. Likewise, by prior addition of LU 52396 to thapsigargin-stimulated Jurkat T cells, a dose-dependent inhibition of Ca2+ signals was achieved. The IC50 value of LU 52396 for both agonists was about 5 microM. LU 52396 also inhibited Jurkat T cell proliferation, but showed cytotoxic effects at concentrations > 50 microM. Our data indicate that, in contrast to the other Ca2+ antagonists SK-F 96365, econazole, nitrendipine and ZnCl2, LU 52396 recognized the channel for capacitative Ca2+ entry only when intracellular Ca2+ was low and the channel was in its closed state.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Piperazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Complexo CD3 , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Ativação Linfocitária
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