RESUMEN
BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of ß-amyloid peptide (Aß) of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP). METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aß40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y), whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Retículo Endoplásmico/metabolismo , Ácido Láctico/farmacología , Chaperonas Moleculares/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
DNA fragmentation factors (DFF) form protein complexes consisting of nuclease DFF40/CAD and inhibitory chaperon DFF45/ICAD. Although activated caspase-3 has been shown to cleave DFF complexes with the release of active DFF40 and DNA fragmentation, the organ-specific mechanisms of DFF turnover during liver injury accompanied by massive apoptosis are unclear. In this study, we investigated hepatic profile of DFF40-immunopositive proteins in two models of liver injury in rats: acute ischemia/reperfusion (I/R) and chronic alcohol administration. We show that DFF40-like proteins occur in intact rat liver mainly as a 52kDa protein. Hepatic I/R-induced caspase-3 activation and a time-dependent accumulation of DFF40-positive protein fragments (40 and 20kDa), most likely via specific caspase-3 cleavage as evidenced by in vitro digestion of intact liver tissue with recombinant caspase-3. In addition, immunoprecipitation with DFF40 followed by Western blot with active caspase-3 antibody revealed the presence of active caspase-3 in DFF40-immunopositive 20kDa proteins. Chronic alcohol administration in rats also resulted in a dose-dependent fragmentation of DFF40 proteins similar to I/R injury. Collectively, these data demonstrate that DFF40 immunopositive proteins exist in the liver as distinct, tissue-specific molecular forms that may be processed by caspase-3 during both acute and chronic liver injury.
Asunto(s)
Caspasa 3/metabolismo , Desoxirribonucleasas/metabolismo , Hepatopatías/enzimología , Hígado/enzimología , Enfermedad Aguda , Animales , Enfermedad Crónica , Desoxirribonucleasas/análisis , Activación Enzimática , Hígado/irrigación sanguínea , Hepatopatías Alcohólicas/enzimología , Proteínas de Unión a Poli-ADP-Ribosa , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/enzimologíaRESUMEN
This study characterizes the receptor binding and functional effects of CVT-3619 [2-{6-[((1R,2R)-2-hydroxycyclopentyl)-amino]purin-9-yl}(4S,5S,2R,3R)-5-[(2-fluorophenylthio)methyl]-oxolane-3,4-diol], a novel N(6)-5' -substituted adenosine analog and A(1) -adenosine receptor (A(1) AdoR) agonist, on rat epididymal and inguinal adipocytes and on the isolated heart and compares these effects with those caused by the full agonist N(6) -cyclopentyladenosine (CPA). In addition, the hypothesis that adipocyte A(1)AdoR are a heterogeneous population with regard to their affinities for ligands was tested. CVT-3619 was 10-100-fold selective for A(1)AdoR versus other AdoR and bound to adipocyte membranes with high (K(H) = 14 nM) and low (K = 5.4 microM) affinities. CVT-3619 reduced cyclic AMP content and release of nonesterified fatty acids from epididymal adipocytes with IC(50) values of 6 and 44 nM, respectively. CVT-3619 was a partial agonist relative to CPA to reduce lipolysis in epididymal and inguinal adipocytes. CVT-3619 did not change atrial rate in rat heart and caused a small (6-ms) prolongation of the stimulus-to-His bundle interval without causing atrioventricular block in guinea pig heart (effects mediated by A(1)AdoR), whereas CPA caused atrioventricular block and near cessation of atrial electrical activity. CVT-3619 increased coronary conductance (effect mediated by A(2A)AdoR) only at concentrations > or =10 microM. Rat epididymal adipocyte A(1)AdoR had similar affinities for the antagonist 8-cyclopentyl-1,3-dipropylxanthine in the presence of three dissimilar A AdoR agonists (2-chloro-N(6) -cyclopentyladenosine, N(6) -sulfophenyladenosine, and N-5' -ethylcarboxamidoadenosine) as determined by Schild analysis. It was concluded that rat epididymal adipocyte A(1)AdoR are a homogeneous receptor population with regard to affinities for ligands and that CVT-3619 is a partial agonist with selectivity for A(1)AdoR and inhibition of lipolysis.
Asunto(s)
Agonistas del Receptor de Adenosina A1 , Adenosina/análogos & derivados , Adipocitos/efectos de los fármacos , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A1 , Adipocitos/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Ácidos Grasos no Esterificados/metabolismo , Femenino , Cobayas , Sistema de Conducción Cardíaco/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Masculino , Miocardio/metabolismo , Unión Proteica , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-DawleyRESUMEN
The purpose of this study was to investigate simultaneously a stress manipulation and an experimental pain manipulation to determine how stress and pain interact to influence negative affectivity. One hundred healthy subjects completed a counterbalanced repeated measure crossover design in which stress (speech task) versus a nonstress control condition (magazine reading) was manipulated. Each session was immediately followed by a 2-minute forehead cold pressor task. Measures of affectivity (Positive Affect Negative Affect Schedule), pain ratings, cardiovascular measures (systolic blood pressure, diastolic blood pressure, heart rate), and salivary cortisol were obtained during each session. Regression analysis showed that the stress manipulation influenced the level of anger and that change in anger predicted post-pain negative affectivity independently of the contribution of maximum pain (model R(2) =.31), with 45% of the total model variance accounted for by change in anger and 17% of the total model variance accounted for by maximum pain intensity. In the nonstress condition only level of pain intensity was an independent predictor of negative affectivity (model R(2) =.16), with 69% of the total model variance accounted for by maximum pain intensity. These results show that stress significantly amplifies post-pain negative mood beyond that accounted for by the level of pain intensity alone.