RESUMO
Caveolae appear in a multitude of processes encompassing growth regulation and trafficking. We demonstrate the abundant presence of ESA/reggie-1/flotillin-2, ATP synthase beta subunit and annexin V in endothelial caveolae by immunopurification of caveolae from vascular endothelial membrane. Five proteins are abundant in a caveolin-1 protein complex, analyzed by sucrose gradient velocity sedimentation following octyl-beta-D-glucopyranoside extraction. Caveolin-1 alpha interacts with caveolin-1beta, caveolin-2, actin, the microsomal form of NADH cytochrome B5 reductase and ESA/reggie-1/flotillin-2 as shown by co-immunoprecipitation. We propose the concept that ATP biosynthesis in caveolae regulates mechanosignaling and is induced by membrane depolarization and a proton gradient. Pressure stimuli and metabolic changes may trigger gene regulation in endothelial cells, involving a nuclear conformer of caveolin-1, shown here with an epitope-specific caveolin-1 antibody, and immediate response of ion channel activity, regulated by ESA/reggie-1/flotillin-2.
Assuntos
Cavéolas/química , Proteínas de Membrana/análise , Animais , Anexina A5/análise , Anexina A5/imunologia , Anexina A5/metabolismo , Cavéolas/imunologia , Cavéolas/metabolismo , Caveolina 1 , Caveolina 2 , Caveolinas/análise , Caveolinas/imunologia , Caveolinas/metabolismo , Linhagem Celular , Citocromo-B(5) Redutase/imunologia , Citocromo-B(5) Redutase/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Imunoprecipitação , Pulmão/citologia , Pulmão/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , ATPases Translocadoras de Prótons/análise , ATPases Translocadoras de Prótons/imunologia , ATPases Translocadoras de Prótons/metabolismo , RatosRESUMO
Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4-10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)-cholesterol (PEG-Chol). Combined results of temperature-dependence analysis and PEG(50)-cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization.
