Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I‐hyaluronan (HA) internalize it using a coated pit pathway [Biochem J, 257:875–884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I‐HA binding to intact LECs at 4°C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I‐HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca+2‐independent HA binding activity increased ∼743%, while the Ca+2‐dependent binding activity was enhanced only ∼46%. Therefore, the Ca+2‐dependent HA binding activity appears not to be intracellular, whereas the Ca+2‐independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I‐HA at 37°C in 10 mM EGT A or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I‐HA in the presence of 10mM Ca125, the amount of cell‐associated radiocctivity increased appoximately 20–50‐fold. However, this additional cell‐associated 125I‐HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10mM EGTA at 4°C. Therefore, the Ca+2‐dependent cell‐associated 125I‐HA had accumulated on the cell surface and had not been internalized. From these studies we conclude the LECs have at least two trypes of specfic HA binding sites. One, the previously characterized HA receptor, is Ca+2‐indepoendent, localized both extracellularly and intracellular, and medicates he efficient binding and subsequent endocytosis of HA using a coated pit pathway. The other newly recognized HA binding activity is Ca+2‐dependent, localized wxtracellularly, and is not responsible for the endocytosis of HA in rat LECs.
- calcium ions
- hyaluronic acid