The endothelial vesicle system in cryofixed frog mesenteric capillaries analysed by ultrathin serial sectioning

Conventional EM sections of chemically fixed capillary endothelial cells reveal numerous apparently free smooth plasmalemmal vesicles. However, the method of ultrathin (less than 150 A) serial sectioning has shown that the smooth vesicle profiles arise merely as a result of the EM thin sectioning of two sets of complex vesicular invaginations from the luminal and abluminal cell surfaces, which end blindly in the cytoplasm. While 50-70% of the total population of vesicular profiles appear to lack connections to the cell surface in conventional (500-700 A thick) EM thin sections less than 1% truly free vesicles can be found by the ultrathin serial section analyses. In the present study it is examined whether similar conclusions apply to endothelial cells which were directly frozen by slam-freezing and subsequently freeze-substituted. The three-dimensional organization of the plasmalemmal vesicular system was analyzed in four series of 19, 18, 13, and 10 ultrathin sections (approximately 110 A thick) of capillaries from frog mesenteries quickly excised from decapitated frogs (Rana pipiens). None of 920 vesicular profiles (diameter 500-1,200 A) which appeared free in individual thin sections of the series represented free vesicles; all profiles either communicated with other vesicles, the cell surface, or in rare cases turned out to be part of cytoplasmic tubular membrane structures. It is concluded that free smooth plasmalemmal vesicles are very rare in rapidly frozen as well as in directly fixed frog capillary endothelium. The volume density of profiles (13-15%), the proportion of apparently free vesicle profiles (70%), and interconnected profiles (20%) were similar to the picture previously found in single EM sections of frog mesenteric capillaries. No transendothelial channels were found in the four series of ultrathin sections of capillaries. However, continuities between the luminal and abluminal cell surfaces were seen in the endothelium of venules. Furthermore, in the ultrathin series of the capillaries, vesicular units belonging to the two sets of invaginations and cytoplasmic tubular membrane structures were in more cases found in very close contact-as fused to share one unit membrane. If this finding is representative for the in vivo situation, it may reflect that the vesicular system represents a highly dynamic system with possibilities for mixing of membranes, cellular traffic of lipid, membrane proteins, and receptors between internal compartments and the cell surfaces, as well as occasional exchange of macromolecules between blood and tissue through rare temporary connections between the two sets of surface invaginations, without actually moving vesicles.