Regulation of aortic endothelial vesicular uptake of cationized ferritin by plasmalemmal binding

Endothelial uptake of cationized ferritin (CF, pI greater than 9.0) was investigated in the rabbit aorta. After in situ perfusion with CF at concentrations less than 0.35 mg/ml in Tyrode's solution, the endothelial plasmalemmal membrane was partially covered by a ferritin monolayer except at some vesicle necks and regions of cell overlap where particles aggregated. At a concentration of 0.5 mg/ml, CF entered all luminal vesicles and bound to the membrane as a uniform layer usually several particles deep. A steady state was reached after 5 s; particle binding and vesicle filling were not affected by perfusion flow rate up to 20 ml/min. The number of particles per loaded vesicle (F/NL) increased with the density of particles at vesicle necks (PDv), suggesting that particle concentration in the vesicle was in equilibrium with that at the neck. At CF concentrations above 1 mg/ml, binding sites at vesicle necks and elsewhere on the membrane became saturated and luminal vesicles became maximally loaded. Under these conditions, F/NL was greater than that expected for equilibrium with the PDv found. Our findings indicate that anionic sites at vesicle necks and cell overlaps are more strongly negatively charged than elsewhere, and suggest that CF vesicle loading is regulated by the degree of binding at the neck and by the relative strength of the attractive forces at the neck and the vesicle interior.