The role of vesicles in the transport of ferritin through frog endothelium.

Abstract

The transport of ferritin molecules by endothelial cell vesicles was quantitatively investigated by EM. Single mesenteric capillaries of pithed frogs were perfused with solutions containing 6.7 g ferritin 100 ml-1 for known periods before fixation in situ with osmium tetroxide. Two series of experiments were carried out: in the 1st series the perfusate contained bovine serum albumin (1.0 g 100 ml-1); in the 2nd series the perfusate contained no protein other than the ferritin. To assess the molecular radius of ferritin in solution, the free diffusion coefficient of ferritin was measured in the presence and absence of albumin. The free diffusion coefficient of ferritin in saline solution (110 mmol l-1) was 0.35 .times. 10-6 cm2 s-1 at 21.degree. C and was not affected by the presence of bovine serum albumin. There is evidently no significant binding of albumin to ferritin in solution and yields a value for the Stokes-Einstein radius of ferritin of 6.1 nm. In all perfusion experiments the percentage of luminal vesicles containing ferritin exceeded the percentage of labeled cytoplasmic vesicles, which in turn exceeded the percentage of labeled abluminal vesicles. Labeling of all vesicle populations was seen after perfusions lasting < 1 s. At this time luminal vesicles were more heavily labeled in the absence of albumin. The labeling of luminal vesicles increased with lengthening perfusion times up to 30-40 s, after which steady levels of labeling were achieved. The rate of rise in luminal labeling and the steady-state levels reached were both greater in the absence of albumin. By contrast cytoplasmic labeling increased above its initial value only after perfusions of > 10 s. In the steady state, labeled cytoplasmic vesicles contained, on average, fewer ferritin molecules than labeled luminal vesicles. This finding is inconsistent with translocation of labeled luminal vesicles across the cell. The early constant labeling of cytoplasmic and abluminal vesicles may be consistent with the existence of vesicular channels. Later cytoplasmic labeling may result from the transient fusion of cytoplasmic vesicles with labeled luminal vesicles for periods long enough to allow mixing of vesicular contents. Albumin may affect vesicular transport by its interaction with the endothelial glycocalyx.