RESUMEN
BACKGROUND/AIMS: Acetaminophen (APAP) effects on intestinal barrier properties are less investigated. APAP may lead to a changed bioavailability of a subsequently administered drug or diet in the body. We investigated the influence of APAP on enterocytic cell membrane properties that are able to modify the net intestinal absorption of administered substances across the Caco-2 barrier model. METHODS: The effect of APAP on cytotoxicity was measured by LDH assay, TER value and cell capacitance label-free using impedance monitoring, membrane permeability by FITC-dextrans, and efflux transporter MDR1 activity by Rh123. APAP levels were determined by HPLC analysis. Cell membrane topography and microvilli were investigated using SEM and intestinal alkaline phosphatase (Alpi) and tight junction protein 1 (TJP1) expression by western blot analysis. RESULTS: APAP changed the apical cell surface, reduced the number of microvilli and protein expression of Alpi as a brush border marker and TJP1, increased the membrane integrity and concurrently decreased cell capacitance over time. In addition, APAP decreased the permeability to small molecules and increased the efflux transporter activity, MDR1. CONCLUSION: APAP alters the Caco-2 cell membrane properties by different mechanisms and reduces the permeability to administered substances. These findings may help to optimize therapeutic implications.
Asunto(s)
Acetaminofén/farmacología , Membrana Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Absorción/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Analgésicos no Narcóticos/farmacología , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Epiteliales/ultraestructura , Proteínas Ligadas a GPI/metabolismo , Humanos , Microscopía Electrónica de Rastreo , Mitocondrias/efectos de los fármacosRESUMEN
The role of the mitogen-activated protein (MAP) kinase phosphatases (MKPs) in light-damaged cells is unclear. Therefore we investigated the involvement of MKP-1 in the regulation of apoptosis and cell survival mediated by MAP kinase pathways in light-damaged human retinal pigment epithelial cells (ARPE-19). Light dose-dependent changes in the expression of MKP-1 and in the phosphorylation status of the MAP kinases, c-Jun-N-terminal kinase (JNK) and p38 were demonstrated. Low light doses up to 2 J cm(-2) led to an upregulation of MKP-1 which resulted in the prevention of cell death by inactivating JNK kinase. However, higher light doses (> or =3 J cm(-2)) significantly reduced MKP-1 protein expression and subsequently led to an increased JNK kinase activity followed by a significant increase in cell death. JNK kinase inactivation by the JNK inhibitor SP600125 significantly reduced light-induced cell death, suggesting that the cytoprotective properties of MKP-1 are mediated mainly by the JNK MAP kinase pathway. Physiological concentrations of ascorbic acid or taurine were seen to prevent apoptosis and cell death in light-damaged ARPE-19 cells by reducing oxidative stress within cells, thus maintaining MKP-1 at high levels, leading to an inactivation of the JNK kinase pathway which resulted in an increased cell viability.
Asunto(s)
Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Epitelio Pigmentado de la Retina/enzimología , Humanos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de la radiaciónRESUMEN
Based on the observation that 5-aminolevulinic acid (ALA) induces the expression of heme oxygenase-1 (HO-1) in cultured melanoma cells, the role of HO-1 on the effectiveness of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) was examined. Transcriptional activation of the HO-1 gene is considered to be an adaptive response to oxidative and cellular stress and confers a protective capacity against cell and tissue injury, which could affect the responsiveness to ALA-PDT. A time-dependent accumulation (0-16 h) of protoporphyrin IX (PPIX) within melanoma cells was seen after incubation with ALA (0.5 mM ALA). Over the same time interval, a significant increase (up to 25-fold) in HO-1 protein expression was observed. Thus, the production and degradation of PPIX (via heme by HO-1) were simultaneously enhanced, leading to a reduced intracellular concentration of the photodynamically active substance PPIX. Diminishing HO-1 activity by the HO-1 inhibitor tin protoporphyrin IX (SnPPIX) significantly enhanced the formation of PPIX up to 1.8 fold. A further strong increase in HO-1 protein expression (up to 128-fold) was seen after ALA-PDT treatment. Induction of HO-1 is an essential step in the 'rescue response' of tumor cells. The pharmacological inhibition of HO-1 activity by SnPPIX leads to a considerable increase in the sensitivity of tumor cells to ALA-PDT treatment. At low radiation doses (0.42 J/cm(2)), the percentages of death cells increased significantly from 7.3+/-1.3% to 43.7+/-6.4%. This effect could be further intensified by cellular depletion of HO-1 mRNA by siRNA. The combination of pharmacological inactivation of HO-1 with gene silencing led to an increase in the death rate of up to 54.1+/-8.6%. The results presented indicate that HO-1 can play a protective role against ALA-PDT mediated cytotoxicity so that a specific inhibition of HO-1 activity and/or expression might be used to increase the efficacy of ALA-based photodynamic therapy.
Asunto(s)
Ácido Aminolevulínico/uso terapéutico , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Fotoquimioterapia , Línea Celular Tumoral , Hemo-Oxigenasa 1/biosíntesis , Humanos , Melanoma/patología , Metaloporfirinas/farmacología , Protoporfirinas/biosíntesis , Protoporfirinas/farmacología , ARN Interferente Pequeño/farmacologíaRESUMEN
Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups.
Asunto(s)
Betaína-Homocisteína S-Metiltransferasa/metabolismo , Carcinoma Hepatocelular/enzimología , Regulación Enzimológica de la Expresión Génica , Neoplasias Hepáticas/enzimología , Equilibrio Hidroelectrolítico , Animales , Betaína/metabolismo , Betaína-Homocisteína S-Metiltransferasa/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Línea Celular Tumoral , Tamaño de la Célula , Proteínas Quinasas Reguladas por Nucleótidos Cíclicos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Concentración Osmolar , Ósmosis , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/metabolismo , Rafinosa/química , Rafinosa/metabolismo , Ratas , Solución Salina Hipertónica/metabolismo , Sarcosina/análogos & derivados , Sarcosina/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Urea/química , Urea/metabolismoRESUMEN
Carotenoids lutein and zeaxanthin are proposed to protect ocular tissues from free-radical damage that can cause cataract and age-related macular degeneration (AMD). They accumulate selectively in the lens and macular region of the retina. Changes in the retinal pigment epithelium are characteristic in AMD. Efficient uptake is essential to study the intracellular effects of carotenoids in cell cultures. For in vitro experiments carotenoids are often dissolved in organic solvents like tetrahydrofuran (THF), dimethylsulfoxide (DMSO) and n-hexane, but difficulties have been associated with these application methods. Recently, O'Sullivan et al. (SM O'Sullivan et al., Br J Nutr 91 (2004) 757) developed a method whereby carotenoids could be delivered to cultured cells without the cytotoxic side effects often observed when organic solvents are used. We modified this method and investigated the effects of different carotenoid-formulations (ethanol/Tween40, methanol/tween40 and acetone/Tween40) on the uptake of lutein and zeaxanthin by differentiated ARPE-19 cells, cell viability and the expression of the "stress" gene HO-1, which is easily induced by a range of stimuli including chemical and physical agents. Micelle formulations prepared with ethanol/Tween40 resulted in the lowest LDH release, the highest carotenoid uptake and the lowest stress response (changes in HO-1 mRNA expression).
Asunto(s)
Células Epiteliales/metabolismo , Luteína/farmacocinética , Micelas , Polisorbatos/farmacología , Xantófilas/farmacocinética , Catarata/tratamiento farmacológico , Catarata/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica , Humanos , L-Lactato Deshidrogenasa/metabolismo , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Retina/citología , Solubilidad , ZeaxantinasRESUMEN
A contribution of intracellular dehydration to insulin resistance has been established in human subjects and in different experimental systems. Here the effect of hyperosmolarity (405 mosmol/l) on insulin-induced mitogen-activated protein (MAP) kinase phosphatase (MKP)-1 expression was studied in H4IIE rat hepatoma cells. Insulin induces robust MKP-1 expression which correlates with a vanadate-sensitive decay of extracellular-signal-regulated kinase (Erk-1/Erk-2) activity. Hyperosmolarity delays MKP-1 accumulation by insulin and this corresponds to impaired MKP-1 synthesis, whereas MKP-1 degradation remains unaffected by hyperosmolarity. Rapamycin, which inhibits signalling downstream from the mammalian target of rapamycin (mTOR) and a peptide inhibiting protein kinase C (PKC) zeta/lambda abolish insulin-induced MKP-1 protein but not mRNA expression, suggesting the involvement of the p70 ribosomal S6 protein kinase (p70S6-kinase) and/or the eukaryotic initiation factor 4E-binding proteins (4E-BPs) as well as atypical PKCs in MKP-1 translation. Hyperosmolarity induces sustained suppression of p70S6-kinase and 4E-BP1 hyperphosphorylation by insulin, whereas insulin-induced tyrosine phosphorylation of the insulin receptor (IR) beta subunit and the IR substrates IRS1 and IRS2, recruitment of the phosphoinositide 3-kinase (PI 3-kinase) regulatory subunit p85 to the receptor substrates as well as PI 3-kinase activation, and Ser-473 phosphorylation of protein kinase B and Thr-410/403 phosphorylation of PKC zeta/lambda are largely unaffected under hyperosmotic conditions. The hyperosmotic impairment of both, MKP-1 expression and p70S6-kinase hyperphosphorylation by insulin is insensitive to K(2)CrO(4), calyculin A and vanadate, and inhibition of the Erk-1/Erk-2 and p38 pathways. The suppression of MKP-1 may further contribute to insulin resistance under dehydrating conditions by allowing unbalanced MAP kinase activation.