IMMUNOISOLATION AND PARTIAL CHARACTERIZATION OF ENDOTHELIAL PLASMALEMMAL VESICLES (CAVEOLAE)

Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginati ons and associated vesicles of regular size and shape found in most ma mmalian cell types. They are particularly numerous in the continuous e ndothelium of certain microvascular beds (e.g., heart, lung, and muscl es) in which they have been identified as transcytotic vesicular carri ers. Their chemistry and function have been extensively studied in the last years by various means, including several attempts to isolate th em by cell fractionation from different cell types. The methods so far used rely on nonspecific physical parameters of the caveolae and thei r membrane (e.g., size-specific gravity and solubility in detergents) which do not rule out contamination from other membrane sources, espec ially the plasmalemma proper. We report here a different method for th e isolation of PVs from plasmalemmal fragments obtained by a silica-co ating procedure from the rat lung vasculature. The method includes son ication and notation of a mixed vesicle fraction, as the first step, f ollowed by specific immunoisolation of PVs on anticaveolin-coated magn etic microspheres, as the second step. The mixed vesicle fraction is t hereby resolved into a bound subfraction (B), which consists primarily of PVs or caveolae, and a nonbound subfraction (NB) enriched in vesic les derived from the plasmalemma proper. The results so far obtained i ndicate that some specific endothelial membrane proteins (e.g., thromb omodulin, functional thrombin receptor) are distributed about evenly b etween the B and NE subfractions, whereas others are restricted to the NE subfraction (e.g., angiotensin converting enzyme, podocalyxin). Gl ycoproteins distribute unevenly between the two subfractions and antig ens involved in signal transduction [e.g., annexin II, protein kinase C alpha, the G alpha subunits of heterotrimeric G proteins (alpha s, a lpha q, alpha i2, alpha i3), small GTP-binding proteins, endothelial n itric oxide synthase, and nonreceptor protein kinase c-src] are concen trated in the NE (plasmalemma proper-enriched) subfraction rather than in the caveolae of the B subfraction. Additional work should show whe ther discrepancies between our findings and those already recorded in the literature represent inadequate fractionation techniques or are ac counted for by chemical differentiation of caveolae from one cell type to another.