Plasmalemmal vesicles and the effects of sterol-binding agents in rabbit aortic endothelium

The rims of vesicle openings in capillary endothelium are reported to be highly sensitive to perturbation by the sterol probe filipin when this agent is administered by perfusion at 50 microM concentration in aldehyde fixative for 10 min. If, as supposed, this specific rim response reflects functionally significant aspects of membrane organization at the vesicle-plasmalemma boundary, then it would be expected to be a reproducible and universal feature of endothelial vesicles in general. In the present study, we have investigated in detail the response to filipin of rabbit and rat aortic endothelial plasmalemma. First, the effect of standard filipin treatment (150 microM, 3-20 h) was examined by freeze-fracture. The extensive generalized response observed, taken together with the effects of filipin seen by fluorescence microscopy of stripped aortic endothelial sheets, and the marked corrugation of the plasmalemma observed by freeze-fracture after tomatin treatment, indicate that substantial amounts of cholesterol are present in the membrane. Exposure of rabbit aortic endothelium to 50 microM filipin for 10, 20, 40 and 60 min enabled the progressive appearance of filipin deformations in the plasmalemma to be traced. Even at the very earliest stages of the response, there was no preferential association of filipin deformations with endothelial vesicle rims. Similar experiments in the rat gave the same result. Specific sensitivity of endothelial vesicle rims would, therefore, seem to be either non-universal in occurrence or non-reproducible, and so is unlikely to reflect any membrane property of fundamental significance to endothelial vesicle function.