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1.
Mol Cell ; 84(10): 1886-1903.e10, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38688280

RESUMO

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.


Assuntos
Neoplasias Hematológicas , Proteínas Musculares , Mutação , Fosfoproteínas , Fatores de Processamento de RNA , Animais , Humanos , Camundongos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/metabolismo , Hematopoese/genética , Íntrons , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo
2.
IUBMB Life ; 76(8): 505-522, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38391119

RESUMO

The amide proteogenic amino acids, asparagine and glutamine, are two of the twenty amino acids used in translation by all known life. The aminoacyl-tRNA synthetases for asparagine and glutamine, asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase, evolved after the split in the last universal common ancestor of modern organisms. Before that split, life used two-step indirect pathways to synthesize asparagine and glutamine on their cognate tRNAs to form the aminoacyl-tRNA used in translation. These two-step pathways were retained throughout much of the bacterial and archaeal domains of life and eukaryotic organelles. The indirect routes use non-discriminating aminoacyl-tRNA synthetases (non-discriminating aspartyl-tRNA synthetase and non-discriminating glutamyl-tRNA synthetase) to misaminoacylate the tRNA. The misaminoacylated tRNA formed is then transamidated into the amide aminoacyl-tRNA used in protein synthesis by tRNA-dependent amidotransferases (GatCAB and GatDE). The enzymes and tRNAs involved assemble into complexes known as transamidosomes to help maintain translational fidelity. These pathways have evolved to meet the varied cellular needs across a diverse set of organisms, leading to significant variation. In certain bacteria, the indirect pathways may provide a means to adapt to cellular stress by reducing the fidelity of protein synthesis. The retention of these indirect pathways versus acquisition of asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase in lineages likely involves a complex interplay of the competing uses of glutamine and asparagine beyond translation, energetic costs, co-evolution between enzymes and tRNA, and involvement in stress response that await further investigation.


Assuntos
Aminoacil-tRNA Sintetases , Evolução Molecular , Biossíntese de Proteínas , Aminoacil-RNA de Transferência , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/genética , Asparagina/metabolismo , Asparagina/genética , Glutamina/metabolismo , Bactérias/genética , Bactérias/enzimologia , Bactérias/metabolismo , Archaea/genética , Archaea/metabolismo , Archaea/enzimologia , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Amidas/metabolismo , Humanos
4.
J Biol Chem ; 290(35): 21376-92, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26152717

RESUMO

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.


Assuntos
Canais de Cálcio/metabolismo , Endossomos/metabolismo , Células Secretoras de Insulina/metabolismo , NADP/análogos & derivados , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Células Cultivadas , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Knockout , NADP/metabolismo
5.
Blood ; 117(18): 4968-77, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21364192

RESUMO

A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.


Assuntos
Células Endoteliais/metabolismo , NADP/análogos & derivados , Receptores Histamínicos H1/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Bases , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacologia , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ácidos Heptanoicos/farmacologia , Histamina/farmacologia , Humanos , NADP/metabolismo , Antagonistas Nicotínicos/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , RNA Interferente Pequeno/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia
6.
Biochem J ; 441(1): 435-42, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21967541

RESUMO

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.


Assuntos
Plaquetas/metabolismo , NADP/análogos & derivados , Ativação Plaquetária/fisiologia , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Carbolinas/farmacologia , Proteínas de Transporte/metabolismo , Humanos , NADP/metabolismo , Peptídeos/metabolismo , Piperazinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Trombina/farmacologia
7.
Biochem Biophys Res Commun ; 418(2): 353-8, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22274607

RESUMO

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca(2+). cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca(2+). Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca(2+). Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.


Assuntos
Permeabilidade da Membrana Celular , ADP-Ribose Cíclica/análogos & derivados , ADP-Ribose Cíclica/metabolismo , Animais , Transporte Biológico , Cafeína/farmacologia , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , ADP-Ribose Cíclica/síntese química , Células PC12 , Ratos , Rianodina/farmacologia , Ouriços-do-Mar , Tapsigargina/farmacologia
8.
Biochem Biophys Res Commun ; 427(2): 326-9, 2012 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22995315

RESUMO

Evidence suggests that ß-Adrenergic receptor signaling increases heart rate and force through not just cyclic AMP but also the Ca(2+)-releasing second messengers NAADP (nicotinic acid adenine dinucleotide phosphate) and cADPR (cyclic ADP-ribose). Nevertheless, proof of the physiological relevance of these messengers requires direct measurements of their levels in response to receptor stimulation. Here we report that in intact Langendorff-perfused hearts ß-adrenergic stimulation increased both messengers, with NAADP being transient and cADPR being sustained. Both NAADP and cADPR have physiological and therefore pathological relevance by providing alternative drug targets in the ß-adrenergic receptor signaling pathway.


Assuntos
ADP-Ribose Cíclica/metabolismo , Miocárdio/metabolismo , NADP/análogos & derivados , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Cobaias , Coração/efeitos dos fármacos , Técnicas In Vitro , NADP/metabolismo , Transdução de Sinais
9.
J Biol Chem ; 285(24): 18262-9, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20400502

RESUMO

Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca(2+) from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn(2+) quenching of luminal fura-2 and (45)Ca(2+) to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca(2+). We propose a new model for acrosome reaction signaling in which Ca(2+) influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca(2+) to drive acrosomal exocytosis.


Assuntos
Reação Acrossômica , NADP/análogos & derivados , Espermatozoides/metabolismo , Acrossomo/metabolismo , Animais , Cálcio/química , Cátions , Exocitose , Feminino , Fura-2/química , Masculino , Manganês/química , NADP/química , Ligação Proteica , Ouriços-do-Mar , Transdução de Sinais
10.
Nat Chem Biol ; 5(4): 220-6, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19234453

RESUMO

Research into the biological role of the Ca(2+)-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca(2+) increases in mouse pancreatic beta cells.


Assuntos
NADP/análogos & derivados , Animais , Carbolinas/química , Carbolinas/farmacologia , ADP-Ribose Cíclica/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Modelos Moleculares , Estrutura Molecular , NADP/química , NADP/metabolismo , Óvulo/química , Piperazinas/química , Piperazinas/farmacologia , Ouriços-do-Mar , Bibliotecas de Moléculas Pequenas
11.
Sci Rep ; 11(1): 7193, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785830

RESUMO

Some studies report that obesity is associated with more severe symptoms following SARS-CoV-2 infection and worse COVID-19 outcomes, however many other studies have not reproduced these findings. Therefore, it is uncertain whether obesity is in fact associated with worse COVID-19 outcomes compared to non-obese individuals. We conducted a systematic search of PubMed (including MEDLINE) and Google Scholar on May 18, 2020 to identify published studies on COVID-19 outcomes in non-obese and obese patients, covering studies published during the first 6 months of the pandemic. Meta-analyses with random effects modeling was used to determine unadjusted odds ratios (OR) and 95% confidence intervals (CI) for various COVID-19 outcomes in obese versus non-obese patients. By quantitative analyses of 22 studies from 7 countries in North America, Europe, and Asia, we found that obesity is associated with an increased likelihood of presenting with more severe COVID-19 symptoms (OR 3.03, 95% CI 1.45-6.28, P = 0.003; 4 studies, n = 974), developing acute respiratory distress syndrome (ARDS; OR 2.89, 95% CI 1.14-7.34, P = 0.025; 2 studies, n = 96), requiring hospitalization (OR 1.68, 95% CI 1.14-1.59, P < 0.001; 4 studies, n = 6611), being admitted to an intensive care unit (ICU; OR 1.35, 95% CI 1.15-1.65, P = 0.001; 9 studies, n = 5298), and undergoing invasive mechanical ventilation (IMV; OR 1.76, 95% CI 1.29-2.40, P < 0.001; 7 studies, n = 1558) compared to non-obese patients. However, obese patients had similar likelihoods of death from COVID-19 as non-obese patients (OR 0.96, 95% CI 0.74-1.25, P = 0.750; 9 studies, n = 20,597). Collectively, these data from the first 6 months of the pandemic suggested that obesity is associated with a more severe COVID-19 disease course but may not be associated with increased mortality.


Assuntos
COVID-19/etiologia , COVID-19/terapia , Obesidade/epidemiologia , Ásia/epidemiologia , COVID-19/epidemiologia , COVID-19/mortalidade , Comorbidade , Europa (Continente)/epidemiologia , Hospitalização/estatística & dados numéricos , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Mortalidade , América do Norte/epidemiologia , Razão de Chances , Respiração Artificial , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/virologia
12.
J Biol Chem ; 284(50): 34930-4, 2009 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19826006

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca(2+) release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.


Assuntos
Carbolinas/metabolismo , NADP/análogos & derivados , Piperazinas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sítios de Ligação , Bioensaio/métodos , Cálcio/metabolismo , Carbolinas/química , Estrutura Molecular , NADP/antagonistas & inibidores , Oócitos/citologia , Oócitos/metabolismo , Piperazinas/química , Ensaio Radioligante , Receptores de Superfície Celular/genética , Ouriços-do-Mar
13.
Biochem J ; 422(3): 503-12, 2009 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-19548879

RESUMO

NAADP (nicotinic acid-adenine dinucleotide phosphate) is an unusual second messenger thought to mobilize acidic Ca(2+) stores, such as lysosomes or lysosome-like organelles, that are functionally coupled to the ER (endoplasmic reticulum). Although NAADP-sensitive Ca(2+) stores have been described in neurons, the physiological cues that recruit them are not known. Here we show that in both hippocampal neurons and glia, extracellular application of glutamate, in the absence of external Ca(2+), evoked cytosolic Ca(2+) signals that were inhibited by preventing organelle acidification or following osmotic bursting of lysosomes. The sensitivity of both cell types to glutamate correlated well with lysosomal Ca(2+) content. However, interfering with acidic compartments was largely without effect on the Ca(2+) content of the ER or Ca(2+) signals in response to ATP. Glutamate but not ATP elevated cellular NAADP levels. Our results provide evidence for the agonist-specific recruitment of NAADP-sensitive Ca(2+) stores by glutamate. This links the actions of NAADP to a major neurotransmitter in the brain.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Ácido Glutâmico/farmacologia , NADP/análogos & derivados , Animais , Células Cultivadas , Feminino , Hipocampo/citologia , Técnicas In Vitro , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , NADP/metabolismo , Neuroglia/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar
14.
Cell Calcium ; 43(6): 531-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17935780

RESUMO

NAADP (nicotinic acid adenine dinucleotide phosphate) is a recently discovered second messenger, and as such, we have much yet to learn about its functions in health and disease. A bottleneck in this basic research is due to NAADP, like all second messengers, being charged to prevent it from leaking out of cells. This makes for effective biology, but imposes difficulties in experiments, as it must be injected, loaded via liposomes, or electroporated, techniques that are highly technically demanding and are possible only in certain single cell preparations. For the better understood second messenger inositol 1,4,5-trisphosphate, great success has been obtained with cell-permeant derivatives where the charged groups are masked through esterification. We now report NAADP-AM as a cell-permeant analogue of NAADP that is taken up into cells and induces NAADP-mediated Ca(2+) signalling. NAADP-AM is a powerful chemical tool that will be of enormous biological utility in a wide range of systems and will greatly facilitate research into the role of NAADP in health and disease.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , NADP/análogos & derivados , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Compostos de Anilina , Animais , Bioquímica/métodos , Sinalização do Cálcio/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Estabilidade de Medicamentos , Corantes Fluorescentes , Cobaias , Masculino , Biologia Molecular/métodos , Estrutura Molecular , NADP/síntese química , NADP/metabolismo , NADP/farmacocinética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Farmacologia/métodos , Ratos , Ratos Wistar , Ouriços-do-Mar , Sistemas do Segundo Mensageiro/fisiologia , Coloração e Rotulagem , Xantenos
15.
Curr Biol ; 15(9): 874-8, 2005 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-15886108

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.


Assuntos
Acetilcolina/farmacologia , Sinalização do Cálcio/fisiologia , Colecistocinina/farmacologia , ADP-Ribose Cíclica/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Acetilcolina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Colecistocinina/metabolismo , Fluorescência , Masculino , Camundongos , Pâncreas/citologia , Ensaio Radioligante , Fatores de Tempo
16.
J Cell Biol ; 200(6): 789-805, 2013 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-23479744

RESUMO

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca(2+) stores that release Ca(2+) in response to the second messengers IP3 and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca(2+) released from acidic organelles by NAADP subsequently recruits IP3 or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca(2+) oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca(2+) released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca(2+)-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca(2+) chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca(2+) at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca(2+) oscillations and waves.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Lytechinus/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Lytechinus/citologia , NADP/análogos & derivados , NADP/biossíntese
17.
FEBS Lett ; 585(14): 2300-6, 2011 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-21664355

RESUMO

Intracellular Ca(2+) signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca(2+) in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca(2+) responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , NADP/análogos & derivados , Animais , Astrócitos/citologia , Bradicinina/metabolismo , Células Cultivadas , Quelantes/metabolismo , Ácido Egtázico/metabolismo , Endotelina-1/metabolismo , NADP/metabolismo , Ratos , Ratos Sprague-Dawley
18.
Anal Biochem ; 371(1): 26-36, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17919448

RESUMO

The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.


Assuntos
Sinalização do Cálcio/fisiologia , NADP/análogos & derivados , Animais , Cálcio/metabolismo , Extratos Celulares/análise , Quelantes/farmacologia , Cromatografia Líquida de Alta Pressão , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Embrião não Mamífero , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos , Microinjeções , Modelos Biológicos , NADP/análise , NADP/isolamento & purificação , NADP/metabolismo , Oócitos/metabolismo , Oxidantes/farmacologia , Pâncreas Exócrino/citologia , Percloratos/farmacologia , Radioisótopos de Fósforo/metabolismo , Ligação Proteica , Ensaio Radioligante , Ouriços-do-Mar/citologia , Ouriços-do-Mar/embriologia , Sistemas do Segundo Mensageiro , Espermatozoides/metabolismo , Fatores de Tempo , Titulometria
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