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Labeling of vesicles in frog rana temporaria endothelial cells with ferritin

Labeling of vesicles in frog rana temporaria endothelial cells with ferritin

Journal of Physiology (Cambridge) 296: 97-112

The labelling of endothelial cell vesicles with ferritin was investigated by EM. Single capillaries in the frog mesentery were perfused with solutions of known concentrations of ferritin for known periods before fixing the tissue in situ by superfusion with osmium tetroxide. At 17.degree. C, the percentage of lumenal vesicles labelled with ferritin increased as the period of perfusion was increased up to 16 s prior to fixation. When perfusions were longer than 16 s, the percentage of vesicles labelled with ferritin remained fairly constant at 70%. At 3.degree. C, no more than 25% of the lumenal vesicles were labelled during the first 30 s. After correcting the data for losses of ferritin due to sectioning, the distribution of ferritin molecules in the lumenal vesicles was consistent with a Poisson distribution. After perfusions of 16 s or longer, the number of ferritin molecules per labelled vesicle was roughly 3-4 times less than would be predicted from the lumenal concentration. At all times there was a gradient of vesicles labelled with ferritin across the endothelial cells, i.e., the percentage of lumenal vesicles labelled was greater than that for cytoplasmic vesicles which in turn was greater than that for vesicles at the ablumenal surface. Whereas the labelling of lumenal vesicles increased from zero time up to 16-20 s, the main increase in labelling of cytoplasmic vesicles occurred between 10 and 20 s. There is a major diffusion barrier to ferritin molecules either close to the endothelial cell surface or across the necks of the lumenal vesicles. Ferritin molecules do not have access to vesicles during the latter part of their residence at the lumenal surface.

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