RESUMEN
Kinetic rate constants (k(GSH)) for the reaction of compounds acting as Michael acceptors with glutathione (GSH) were modelled by quantum chemical transition-state calculations at the B3LYP/6-31G** and B3LYP/TZVP level. The data set included α, ß-unsaturated aldehydes, ketones and esters, with double bonds and triple bonds, linear and cyclic systems, both with and without substituents in the α-position. Predicted values for k(GSH) were found to be in good agreement with experimental k(GSH) values. Factors affecting rate constants have been elucidated, especially solvent effects and the influence of steric hindrance. Solvent effects were examined by adding explicit solvent molecules to the system and by using a polarizable continuum solvent model. Detailed analysis of transition-state energies shows that the reaction is reversible. The reactive enolic intermediate plays an important role in Michael addition to GSH, while the subsequent keto-enol-tautomerism is not rate limiting.
Asunto(s)
Glutatión/química , Aldehídos/química , Ésteres/química , Cetonas/química , Cinética , Modelos Químicos , Transición de Fase , Relación Estructura-Actividad CuantitativaRESUMEN
Although many properties of polyatomic metal clusters have been rationalized by an electron shell model resembling that used for free atoms, it remained unclear how reliable this analogy is with respect to the angular momentum eigenstate character of the electronic wave functions. We studied free size-selected negatively charged clusters of sodium atoms (Nan-) of approximately spherical shape (n = 19, 40, 55, 58, 147) by angle-resolved photoelectron spectroscopy over a broad range of photon energies (1.5 to 5 electron volts). Highly anisotropic, state- and energy-dependent angular distributions emerged for all sizes. Well-defined classes of energy dependence related to the approximate angular momenta of the bound-state orbitals indicate that the overall character of the valence electron wave functions is not appreciably influenced by the interaction with the ion background. The measured distributions nevertheless deviate strongly from the predictions of single-electron models, hinting at a distinct role of correlated multielectron effects in the photoemission process.
RESUMEN
Angiotensin II (ANG II) and nitric oxide (NO) have contrasting vascular effects, yet both sustain inflammatory responses. We investigated the impact of ANG II on lipopolysaccharide (LPS)/interferon-gamma (IFN)-induced NO production in cultured rat mesangial cells (MCs). LPS/IFN-induced nitrite production, the inducible form of nitric oxide synthase (NOS-2) mRNA, and protein expression were dose dependently inhibited by ANG II on coincubation, which was abolished on ANG II type 2 (AT(2)) receptor blockade by PD-123319. Homology-based RT-PCR verified the presence of AT(1A), AT(1B), and AT(2) receptors. To shift the AT receptor expression toward the type 1 receptor, two sets of experiments were performed: LPS/IFN preincubation for 24 h was followed by 8-h coincubation with ANG II; or during 24-h coincubation of LPS/IFN and ANG II, dexamethasone was added for the last 6-h period. Both led to an amplified overall expression of NOS-2 protein and NO production that was inhibitable by actinomycin D in the first setup. Induced NO production was enhanced via the AT(1) receptor; however, it was diminished via the AT(2) receptor. In conclusion, induced NO production is negatively controlled by the AT(2), whereas AT(1) receptor stimulation enhanced NO synthesis in MCs. The overall NO availability depended on the onset of the inflammatory stimuli with respect to ANG II exposure and the available AT receptors.
Asunto(s)
Angiotensina II/metabolismo , Mesangio Glomerular/metabolismo , Óxido Nítrico/biosíntesis , Receptores de Angiotensina/metabolismo , Animales , Antiinflamatorios/farmacología , Northern Blotting , Western Blotting , Células Cultivadas , Sondas de ADN/metabolismo , Dexametasona/farmacología , Mesangio Glomerular/citología , Interferones/farmacología , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/efectos de los fármacos , Receptores de Angiotensina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cytokine-induced nitric oxide (NO) is produced on glomerular inflammation. Glomerular injury and thrombocyte aggregation result in the release of nucleotides, which may regulate induced NO synthesis in cultured rat mesangial cells (MCs). ATP (10(-3) M) inhibited 24-h nitrite production induced by lipopolysaccharide (LPS, 10 microg/ml)/interferon-gamma (IFN-gamma, 100 U/ml) by 48.2 +/- 6. 3%, as well as induction of inducible NOS (iNOS) protein and mRNA. Also, coincubation with either 10(-4) M of UTP, ATP, or ATPgammaS inhibited LPS/IFN-gamma-induced nitrite production by 29.9 +/- 5.8, 36.4 +/- 4.3, and 50.3 +/- 6.5%, respectively, indicating involvement of purinergic P2Y2 receptors. Correspondingly, cultured MCs expressed P2Y2 receptor mRNA. Agonists for other purinergic receptors [alpha,beta-methylene-ATP, 3'-O-(4-benzoyl)-benzoyl-ATP, 2-methylthio-ATP, ADP, UDP, adenosine] were ineffective. Treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 10(-8) M) reproduced the inhibitory effect of ATP on iNOS protein expression and nitrite inhibition (by 46.6 +/- 10. 4%). The effect of ATP or PMA was reversed by the PKC inhibitors Ro-31-8220 (10(-8) M) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10(-5) M), indicating that suppression of iNOS is mediated via activation of PKC through stimulated P2Y2 receptors. In conclusion, the release of purine mediators may play a critical role for iNOS expression and synthesis of NO during glomerular inflammatory disorders.
Asunto(s)
Adenosina Trifosfato/farmacología , Mesangio Glomerular/fisiología , Interferón gamma/farmacología , Óxido Nítrico Sintasa/biosíntesis , Receptores Purinérgicos P2/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenosina Trifosfato/análogos & derivados , Animales , Células Cultivadas , Activación Enzimática , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/enzimología , Indoles/farmacología , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Biosíntesis de Proteínas/efectos de los fármacos , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y2 , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , omega-N-Metilarginina/farmacologíaRESUMEN
The effects of cytokines, lipopolysaccharide (LPS), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), and pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappa B) activation, on inducible nitric oxide synthase (iNOS) expression were studied in the medullary thick ascending limb of Henle's loop cell line ST-1. LPS + interferon-gamma (IF-gamma) promoted a time-dependent increase in nitrite (a NO metabolite) and iNOS mRNA and the appearance of NF-kappa B p50 and p65 in nuclear protein extracts. Actinomycin D but not cycloheximide prevented the LPS + IF-gamma induction of iNOS mRNA and NO synthesis, indicating that iNOS transcriptional activation by LPS + IF-gamma does not require newly synthesized proteins. PDTC inhibited the LPS + IF-gamma induction of NO, iNOS mRNA, and the appearance of NF-kappa B in nuclear protein extracts, suggesting that NF-kappa B mobilization and trans-activation of the iNOS gene mediates this induction. In contrast to other cell types, cycloheximide did not alter iNOS mRNA stability, and 8-BrcAMP did not alter basal or LPS+IF-gamma induced NO production in ST-1 cells.
Asunto(s)
Asa de la Nefrona/enzimología , FN-kappa B/fisiología , Óxido Nítrico Sintasa/biosíntesis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Citocinas/farmacología , Inducción Enzimática , Expresión Génica , Humanos , Lipopolisacáridos/farmacología , Asa de la Nefrona/citología , Ratones , Datos de Secuencia Molecular , Mucoproteínas/genética , Mucoproteínas/metabolismo , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Sondas de Oligonucleótidos/genética , Pirrolidinas/farmacología , Ratas , Tiocarbamatos/farmacología , UromodulinaRESUMEN
The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.