RESUMEN
Gastrointestinal dysfunction is common in diabetic patients. In genetic (nonobese diabetic) and toxin-elicited (streptozotocin) models of diabetes in mice, we demonstrate defects in gastric emptying and nonadrenergic, noncholinergic relaxation of pyloric muscle, which resemble defects in mice harboring a deletion of the neuronal nitric oxide synthase gene (nNOS). The diabetic mice manifest pronounced reduction in pyloric nNOS protein and mRNA. The decline of nNOS in diabetic mice does not result from loss of myenteric neurons. nNOS expression and pyloric function are restored to normal levels by insulin treatment. Thus diabetic gastropathy in mice reflects an insulin-sensitive reversible loss of nNOS. In diabetic animals, delayed gastric emptying can be reversed with a phosphodiesterase inhibitor, sildenafil. These findings have implications for novel therapeutic approaches and may clarify the etiology of diabetic gastropathy.
Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/enzimología , Insulina/farmacología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Gastropatías/tratamiento farmacológico , Animales , Complicaciones de la Diabetes , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/enzimología , Vaciamiento Gástrico/efectos de los fármacos , Vaciamiento Gástrico/fisiología , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa de Tipo I , ARN Mensajero/genética , ARN Mensajero/metabolismo , Gastropatías/enzimología , Gastropatías/etiologíaRESUMEN
While molecular mechanisms for iron entry and storage within cells have been elucidated, no system to mediate iron efflux has been heretofore identified. We now describe an ATP requiring iron transporter in mammalian cells. (55)Fe is transported into microsomal vesicles in a Mg-ATP-dependent fashion. The transporter is specific for ferrous iron, is temperature- and time-dependent, and detected only with hydrolyzable nucleotides. It differs from all known ATPases and appears to be a P-type ATPase. The Fe-ATPase is localized together with heme oxygenase-1 to microsomal membranes with both proteins greatly enriched in the spleen. Iron treatment markedly induces ATP-dependent iron transport in RAW 264.7 macrophage cells with an initial phase that is resistant to cycloheximide and actinomycin D and a later phase that is inhibited by these agents. Iron release, elicited in intact rats by glycerol-induced rhabdomyolysis, induces ATP-dependent iron transport in the kidney. Mice with genomic deletion of heme oxygenase-1 have selective tissue iron accumulation and display augmented ATP-dependent iron transport in those tissues that accumulate iron.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Hierro/metabolismo , Microsomas/enzimología , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Animales , Ácido Ascórbico/farmacología , Encéfalo/metabolismo , Cationes Bivalentes/farmacología , Línea Celular , Células Cultivadas , Inducción Enzimática , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Hierro/farmacología , Riñón/metabolismo , Macrófagos , Proteínas de la Membrana , Ratones , Microsomas Hepáticos/metabolismo , Ratas , Bazo/enzimología , Especificidad por SustratoRESUMEN
Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.
Asunto(s)
Apoptosis/fisiología , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hierro/metabolismo , Piel/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Clonación Molecular , Medio de Cultivo Libre de Suero , Etopósido/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/patología , Eliminación de Gen , Hemo Oxigenasa (Desciclizante)/deficiencia , Hemo-Oxigenasa 1 , Humanos , Proteínas de la Membrana , Ratones , Ratones Noqueados , Proteínas Recombinantes/metabolismo , Piel/citología , Piel/efectos de los fármacos , Estaurosporina/farmacología , TransfecciónRESUMEN
RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Sirolimus/farmacología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Mutación , Fosforilación , Ratas , Receptores de Glicina/metabolismo , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TORRESUMEN
The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1/FRAP/mTOR, the in vivo rapamycin-sensitive target and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. Here, we show that RAFT1 directly phosphorylates p70(S6k), 4E-BP1, and 4E-BP2 and that serum stimulates RAFT1 kinase activity with kinetics similar to those of p70(S6k) and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70(S6k) on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70(S6k) much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70(S6k) is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1.
Asunto(s)
Proteínas Portadoras/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Factor 4E Eucariótico de Iniciación , Proteínas de Choque Térmico/metabolismo , Humanos , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/metabolismo , Mapeo Peptídico , Fosforilación , Fosfotreonina/metabolismo , Polienos/metabolismo , Sirolimus , Serina-Treonina Quinasas TOR , Proteínas de Unión a TacrolimusRESUMEN
The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4- and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.
Asunto(s)
Proteínas Portadoras/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas de Saccharomyces cerevisiae , 1-Fosfatidilinositol 4-Quinasa , Androstadienos/farmacología , Animales , Encéfalo/metabolismo , Proteínas Portadoras/inmunología , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Polienos/metabolismo , Pruebas de Precipitina , Ratas , Sirolimus , Serina-Treonina Quinasas TOR , Proteínas de Unión a Tacrolimus , WortmaninaRESUMEN
We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I] (azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H]BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H]BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H]BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.
Asunto(s)
Marcadores de Afinidad/metabolismo , Canales de Calcio , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cromatografía Líquida de Alta Presión , Inositol 1,4,5-Trifosfato/análogos & derivados , Receptores de Inositol 1,4,5-Trifosfato , Radioisótopos de Yodo , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fotoquímica , Ratas , TritioRESUMEN
High affinity [3H]noscapine binding sites are brain specific, ion insensitive, and present in a variety of species and show strict structure-activity requirements. Among neurotransmitter-related structures, indoleamines and beta-carbolines display highest affinity for [3H]noscapine sites. Noscapine inhibits carbachol-stimulated phosphoinositide turnover in guinea pig and rat brain slices, with structural analogs possessing similar relative potencies for binding to [3H]noscapine binding sites and inhibiting phosphoinositide turnover. Noscapine and its derivatives also markedly enhance the ability of forskolin to augment cAMP levels in brain slices, with relative potencies paralleling affinities for noscapine binding sites.
Asunto(s)
Adenilil Ciclasas/metabolismo , Encéfalo/metabolismo , Indoles/metabolismo , Noscapina/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Carbacol/farmacología , Colforsina/farmacología , Cricetinae , Cobayas , Técnicas In Vitro , Ratones , Ratas , Sistemas de Mensajero Secundario , Relación Estructura-ActividadRESUMEN
We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.
Asunto(s)
Fosfatos de Inositol/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares , Marcadores de Afinidad , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Cationes Bivalentes , Cromatografía de Afinidad , Cinética , Masculino , Fotoquímica , Ratas , Ratas Endogámicas , Especificidad por SustratoRESUMEN
High-affinity, membrane-associated inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6) binding proteins were solubilized and isolated utilizing a heparin-agarose resin followed by an IP4 affinity resin. The IP6 receptor comprises a protein complex of 115-, 105-, and 50-kDa subunits, all of which comigrate under native conditions. The Kd of the receptor for IP6 is 12 nM, whereas inositol 1,3,4,5,6-pentakisphosphate (IP5), IP4, and inositol 1,4,5-trisphosphate (IP3) are 50%, 30%, and 15%, respectively, as potent. Two protein complexes copurify with the IP4 receptor fraction. A 182/123-kDa complex elutes first from the affinity column followed by a 174/84-kDa protein complex, which elutes at higher salt. Both complexes show high affinity for IP4 (Kd = 3-4 nM). IP5, IP6, and IP3 display approximately 25%, 10%, and 0.1%, respectively, the affinity of IP4. Ligand binding to IP6 and IP4 receptors is inhibited 50% by heparin at 0.1 microgram/ml. IP4 receptor proteins are stoichiometrically phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, whereas negligible phosphorylation is observed for the IP6 receptor.