RESUMEN
Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca(2+) channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca(2+) concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca(2+)-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Asunto(s)
Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Calcio/metabolismo , Forma de la Célula , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Corteza Suprarrenal/efectos de los fármacos , Angiotensina II/farmacología , Canales de Calcio Tipo L , Calmodulina/metabolismo , Línea Celular , Forma de la Célula/efectos de los fármacos , Impedancia Eléctrica , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Imagenología Tridimensional , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ouabaína/farmacología , ARN Interferente Pequeño/metabolismo , Tapsigargina/farmacología , Factores de TiempoRESUMEN
The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-ß-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-ß-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [³H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [³H]neutral sterols and [³H]bile acids, whereas all compounds increased plasma HDL-[³H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-ß-HDL formation, which may be required to increase reverse cholesterol transport.
Asunto(s)
Anticolesterolemiantes/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas de Alta Densidad Pre-beta/metabolismo , Amidas , Animales , Ácidos y Sales Biliares/metabolismo , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Cricetinae , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Ésteres , Lipoproteínas de Alta Densidad Pre-beta/sangre , Humanos , Oxazolidinonas/farmacología , Quinolinas/farmacología , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
BACKGROUND: The risk of cardiovascular disease is inversely correlated to level of plasma HDL-c. Moreover, reverse cholesterol transport (RCT) from peripheral tissues to the liver is the most widely accepted mechanism linked to the anti-atherosclerotic activity of HDL. The apolipoprotein A-I (apoA-I) and the ABC transporters play a key role in this process.Adipose tissue constitutes the body's largest pool of free cholesterol. The adipose cell could therefore be regarded as a key factor in cholesterol homeostasis. The present study investigates the capacity of primary cultures of mature human adipocytes to release cholesterol and explores the relationships between apoA-I, ABCA1, and apoE as well as the signaling pathways that could be potentially involved. RESULTS: We demonstrate that apoA-I induces a strong increase in cholesterol release and apoE secretion from adipocytes, whereas it has no transcriptional effect on ABCA1 or apoE genes. Furthermore, brefeldin A (BFA), an intracellular trafficking inhibitor, reduces basal cholesterol and apoE secretion, but does not modify induction by apoA-I. The use of statins also demonstrates that apoA-I stimulated cholesterol release is independent of HMG-CoA reductase activation. CONCLUSION: Our work highlights the fact that adipose tissue, and particularly adipocytes, may largely contribute to RCT via a mechanism specifically regulated within these cells. This further supports the argument that adipose tissue must be regarded as a major factor in the development of cardiovascular diseases, in particular atherosclerosis.
Asunto(s)
Adipocitos/metabolismo , Apolipoproteína A-I/fisiología , Apolipoproteínas E/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adipocitos/efectos de los fármacos , Adulto , Apolipoproteínas E/genética , Aterosclerosis/fisiopatología , Células Cultivadas , AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Persona de Mediana Edad , Sistemas de Mensajero Secundario/efectos de los fármacos , Vías Secretoras/efectos de los fármacos , Grasa Subcutánea/citología , Grasa Subcutánea/efectos de los fármacos , Grasa Subcutánea/metabolismo , Grasa Subcutánea/fisiopatología , Factores de TiempoRESUMEN
OBJECTIVE: The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. METHODS: Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Cachannel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. RESULTS: Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1-5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca channel antagonism, but was blocked by several L-type Cachannel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca channel. CONCLUSION: The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.