Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells |
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Authors: | Trevisi Lucia Pighin Isabella Bazzan Sara Luciani Sisto |
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Affiliation: | Department of Pharmacology and Anaesthesiology, “Egidio Meneghetti”, University of Padua, 35131 Padua, Italy |
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Abstract: | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1-200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K-ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase. |
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Keywords: | bFGF, basic fibroblast growth factor EGFR, epidermal growth factor receptor ERK, extracellular signal-regulated kinase FBS, fetal bovine serum HUVEC, human umbilical vein endothelial cells MβCD, methyl β-cyclodextrin MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PI3K, phosphatidylinositol 3 kinase PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine |
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