Reduction of cell density dependent antigenic properties of intracellular Ca(2+)-regulating membranes by isoflurane in human endothelial cells.

Abstract

An established cell line of human umbilical vein endothelial cells displays a pronounced shift in the distribution of intracellular Ca(2+)-regulating membranes as the cells grow towards confluence. In sparsely populated cultures a linearly oriented punctate pattern of vesicle-like structures is observed similar to the distribution found in many other eucaryotic cells. As the cell population increases, the membranes condense around the nucleus. In completely confluent cultures when the cells cease to proliferate, the antigen is no longer detectable by immunofluorescence; its absence is confirmed by Western blotting experiments. Double-labeling with antitubulin or phalloidin shows that the distribution of microtubules is not related to the distribution of the Ca(2+)-regulating membranes whereas the actin fibers are superimposable onto the linearly oriented punctate vesicle-like structures. If proliferating cells are treated with the volatile anesthetic isoflurane, the Ca(2+)-regulating membranes can no longer be detected with the antibody; however, in Western blots the antigen is still present. Staining is restored after the removal of isoflurane. Such a temporary disappearance of the immunocytochemical staining might be explained by a transient oxidation and subsequent configuration change of the antigen, rendering the epitope inaccessible to the antibody, since a treatment with low concentrations of NaBH4 of cells exposed to isoflurane restores the access of the antigen to the antibody.