ENDOTHELIAL-CELLS REESTABLISH FUNCTIONAL INTEGRITY AFTER REVERSIBLE PERMEABILIZATION

Permeabilization is an important tool in cell biology that allows manipulation of intracellular mechanisms by introduction of probes and regulatory molecules into the cell cytoplasm. We found that incubation of endothelial cells (ECs) with glass beads resulted in nonspecific permeabilization of human and bovine ECs without removal of the cells from monolayer culture. This poration of the plasma membrane allowed the introduction of macromolecules (dextrans less-than-or-equal-to 152 kd and immunoglobulins) as well as small, charged molecules (Lucifer Yellow). We found that nonspecific permeabilization of the EC was transient and defined the conditions under which integrity of the plasma membrane was reestablished. This process was dependent on time, temperature, and the presence of extracellular calcium. We also demonstrated that permeabilized ECs regain functional characteristics. This was defined by a number of criteria, including the ability to rapidly reestablish confluent monolayer morphology, to extrude Lucifer Yellow, to adhere and spread after passage, and to synthesize biologically active molecules after stimulation with a receptor-mediated agonist. Thus, transiently porated ECs loaded with appropriate probes can be used in studies of regulatory mechanisms while remaining in monolayer culture, a condition in which many phenotypic features are similar to in situ endothelium. Furthermore, the porated EC may be a useful model for defining the mechanisms that influence the repair of endothelial plasma membrane injury.